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United States Patent |
5,683,905
|
Capon
,   et al.
|
November 4, 1997
|
Vectors and cell lines capable of correctly splicing exon regions
Abstract
A recombinant DNA vector is provided that expresses exons of genomic DNA
fragments that are inserted into the vector. The vector contains a
promoter and a genomic DNA fragment so characterized and configured that
the vector, upon transcription in a transfected eukaryotic cell culture,
expresses the corresponding RNA segment of the genomic DNA fragment free
of any intron.
Inventors:
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Capon; Daniel J. (San Mateo, CA);
Lawn; Richard M. (San Francisco, CA);
Vehar; Gordon A. (San Carlos, CA);
Wood; William I. (San Mateo, CA)
|
Assignee:
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Genentech, Inc. (South San Francisco, CA)
|
Appl. No.:
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448171 |
Filed:
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May 23, 1995 |
Current U.S. Class: |
435/320.1 |
Intern'l Class: |
C12N 015/63; C12N 005/00 |
Field of Search: |
435/320.1,240.1
|
References Cited
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|
Primary Examiner: Robinson; Douglas W.
Assistant Examiner: Wai; Thanda
Attorney, Agent or Firm: Hasak; Janet E., McNicholas; Janet M.
Parent Case Text
This is a continuation of applications(s) Ser. No. 07/595,481 filed on 09
Oct. 1990 which is a continuation of Ser. No. 07/083,758 filed 07 Aug.
1987, now U.S. Pat. No. 4,965,199 which is a continuation of Ser. No.
06/602,312 filed 20 Apr. 1984, now abandoned, which applications are
incorporated herein by reference and to which applications priority is
claimed under 35 USC .sctn.120.
Claims
We claim:
1. A recombinant DNA vector which expresses exons of genomic DNA fragments
inserted into the vector, which vector comprises: (1) a promoter operably
linked to a splice donor site followed by a splice acceptor site and (2) a
genomic DNA fragment inserted between said splice donor and splice
acceptor sites, wherein the genomic DNA fragment comprises a splice
acceptor site followed by a splice donor site, and wherein said vector,
upon transcription in a transfected eukaryotic cell culture, expresses the
corresponding RNA segment of said genomic DNA fragment free of any intron.
2. The vector of claim 1 wherein the genomic DNA fragment codes for a
polypeptide of interest.
3. The vector of claim 2 wherein the polypeptide is human factor VIII.
4. A eukaryotic cell culture transfected with a vector according to claim
1.
5. pESVDA.
6. A eukaryotic cell culture transfected with a vector according to claim 5
.
Description
FIELD OF THE INVENTION
The present invention relates to human factor VIII, to novel forms and
compositions thereof and particularly to means and methods for the
preparation of functional species of human factor VIII, particularly via
recombinant DNA technology.
The present invention is based in part on the discovery of the DNA sequence
and deduced amino acid sequence of human factor VIII as well as associated
portions of the factor VIII molecule found in our hands to be functional
bioactive moieties. This discovery was enabled by the production of factor
VIII in various forms via the application of recombinant DNA technology,
thus, in turn enabling the production of sufficient quality and quantity
of materials with which to conduct biological testing and prove biological
functionality. Having determined such, it is possible to tailor-make
functional species of factor VIII via genetic manipulation and in vitro
processing, arriving efficiently at hitherto unobtainable commercially
practical amounts of active factor VIII products. This invention is
directed to these associated embodiments in all respects.
The publications and other materials hereof used to illuminate the
background of the invention, and in particular cases, to provide
additional details concerning its practice are incorporated herein by
reference and listed at the end of the specification in the form of a
bibliography.
BACKGROUND OF THE INVENTION
The maintenance of an intact vascular system requires the interaction of a
variety of cells and proteins. Upon injury to the vascular bed, a series
of reactions is initiated in order to prevent fluid loss. The initial
response is the activation of platelets, which adhere to the wound and
undergo a series of reactions. These reactions include the attraction of
other platelets to the site, the release of a number of organic compounds
and proteins, and the formation of a thrombogenic surface for the
activation of the blood coagulation cascade. Through this combined series
of reactions, a platelet plug is formed sealing the wound. The platelet
plug is stabilized by the formation of fibrin threads around the plug
preventing unwanted fluid loss. The platelet plug and fibrin matrix are
subsequently slowly dissolved as the wound is repaired. For a general
review, see (1).
A critical factor in the arrest of bleeding is the activation of the
coagulation cascade in order to stabilize the initial platelet plug. This
system consists of over a dozen interacting proteins present in plasma as
well as released and/or activated cellular proteins (2, 3). Each step in
the cascade involves the activation of a specific inactive (zymogen) form
of a protease to the catalytically active form. By international agreement
(4), each protein of the cascade has been assigned a Roman numeral
designation. The zymogen form of each is represented by the Roman numeral,
while the activated form is represented by the Roman numeral followed by a
subscript "a". The activated form of the protease at each step of the
cascade catalytically activates the protease involved in the subsequent
step in the cascade. In this manner a small initial stimulus resulting in
the activation of a protein at the beginning of the cascade is
catalytically amplified at each step such that the final outcome is the
formation of a burst of thrombin, with the resulting thrombin catalyzed
conversion of the soluble protein fibrinogen into its insoluble form,
fibrin. Fibrin has the property of self-aggregating into threads or fibers
which function to stabilize the platelet plug such that the plug is not
easily dislodged.
FIG. 1 summarizes the current understanding of the interactions of the
proteins involved in blood coagulation. The lack or deficiency of any of
the proteins involved in the cascade would result in a blockage of the
propagation of the initial stimulus for the production of fibrin. In the
middle of the cascade represented in FIG. 1 is a step wherein factor
IX.sub.a initiates the conversion of factor X to the activated form,
factor X.sub.a. Factor VIII (also synonomously referred to as factor
VIIIC) is currently believed to function at this step, in the presence of
phospholipid and calcium ions, as a cofactor; that is, it has no known
function in itself, and is required to enhance the activity of factor IXa.
This step in the cascade is critical since the two most common hemophilia
disorders have been determined to be caused by the decreased functioning
of either factor VIII (hemophilia A or classic hemophilia) or factor IXa
(hemophilia B). Approximately 80 percent of hemophilia disorders are due
to a deficiency of factor VIII. The clinical manifestation in both types
of disorders are the same: a lack of sufficient fibrin formation required
for platelet plug stabilization, resulting in a plug which is easily
dislodged with subsequent rebleeding at the site. The relatively high
frequency of factor VIII and factor IX deficiency when compared with the
other factors in the coagulation cascade is due to their genetic linkage
to the X-chromosome. A single defective allele of the gene for factor VIII
or factor IX results in hemophilia in males, who have only one copy of the
X chromosome. The other coagulation factors are autosomally linked and
generally require the presence of two defective alleles to cause a blood
coagulation disorder--a much less common event. Thus, hemophilia A and B
are by far the most common hereditary blood clotting disorders and they
occur nearly exclusively in males.
Several decades ago the mean age of death of hemophiliacs was 20 years or
younger. Between the early 1950's and the late 1960's, research into the
factor VIII disorder led to the treatment of hemophilia A initially with
whole plasma and, later, with concentrates of factor VIII. The only source
for human factor VIII has been human plasma. One factor contributing to
the expense is the cost associated with obtaining large amounts of usable
plasma. Commercial firms must establish donation centers, reimburse
donors, and maintain the plasma in a frozen state immediately after
donation and through the shipment to the processing plant. The plasma
samples are pooled into lots of over 1000 donors and processed. Due to the
instability of the factor VIII activity, large losses are associated with
the few simple purification procedures utilized to produce the
concentrates (resulting in approximately a 15 percent recovery of
activity). The resulting pharmaceutical products are highly impure, with a
specific activity of 0.5 to 2 factor VIII units per milligram of protein
(one unit of factor VIII activity is by definition the activity present in
one milliliter of plasma). The estimated purity of factor VIII concentrate
is approximately 0.04 percent factor VIII protein by weight. This high
impurity level is associated with a variety of serious complications
including precipitated protein, hepatitis, and possibly the agent
responsible for Acquired Immune Deficiency Syndrome. These disadvantages
of the factor VIII concentrates are due to the instability of the plasma
derived factor VIII, to its low level of purity, and to its derivation
from a pool of multiple donors. This means that should one individual out
of the thousand donors have, for example, hepatitis, the whole lot would
be tainted with the virus. Donors are screened for hepatitis B, but the
concentrates are known to contain both hepatitis A and hepatitis non-A
non-B. Attempts to produce a product of higher purity result in
unacceptably large losses in activity, thereby increasing the cost.
The history of purification of factor VIII illustrates the difficulty in
working with this protein. This difficulty is due in large part to the
instability and trace amounts of factor VIII contained in whole blood. In
the early 1970's, a protein was characterized which was then believed to
be factor VIII (5, 6, 7). This protein was determined to be an aggregate
of a subunit glycoprotein, the subunit demonstrating a molecular weight of
approximately 240,000 daltons as determined by SDS gel electrophoresis.
This subunit aggregated into a heterogeneous population of higher
molecular weight species ranging from between one million and twenty
million daltons. The protein was present in hemophiliac plasma, but
missing in plasma of patients with von Willebrand's disease, an
autosomally transmitted genetic disorder characterized by a prolonged
bleeding time and low levels of factor VIII (8). The theory then proposed
was that this high molecular weight protein, termed von Willebrand factor
(vWF) or factor VIII related antigen (FVIIIRAg), was responsible for the
coagulation defect in both diseases, with the protein being absent in von
Willebrand's disease and somehow non-functional in classic hemophilia
disease states (9). However, it was later observed that under certain
conditions, notably high salt concentrations, the factor VIII activity
could be separated from this protein believed responsible for the activity
of factor VIII (10-20). Under these conditions, the factor VIII coagulant
activity exhibited a molecular weight of 100,000 to 300,000. Since this
time, great effort has concentrated on identifying and characterizing the
protein(s) responsible for the coagulant activity of factor VIII. However,
the availability of but trace amounts of the protein in whole blood
coupled with its instability have hampered such studies.
Efforts to isolate factor VIII protein(s) from natural source, both human
and animal, in varying states of purity, have been reported (21-27, 79).
Because of the above mentioned problems, the possibility exists for the
mistaken identification and subsequent cloning and expression of a
contaminating protein in a factor VIII preparation rather than the factor
VIII protein intended. That this possibility is real is emphasized by the
previously mentioned mistaken identification of von Willebrand protein as
being the factor VIII coagulant protein. Confusion over the identification
of factor VIII-like activity is also a distinct possibility. Either factor
Xa or thrombin would cause a shortening of the clotting time of various
plasmas, including factor VIII deficient plasma, thereby appearing to
exhibit factor VIII-like activity unless the proper controls were
performed. Certain cells are also known to produce activities which can
function in a manner very similar to that expected of factor VIII (28, 29,
30). The latter reference (30) proves that this factor VIII-like activity
is in fact a protein termed tissue factor. The same or similar material
has also been purified from human placenta (31). This protein functions,
in association with the plasma protein factor VII, at the same step as
factor VIII and factor IX.sub.a, resulting in the activation of factor X
to factor X.sub.a.
The burden of proof for expression of a recombinant factor VIII would
therefore rest on the proof of functional expression of what is
unquestionably a factor VIII activity. Even were prior workers to show
that they obtained a full or partial clone encoding all or a portion of
factor VIII, the technical problems in the expression of a recombinant
protein which is four times larger than any other recombinant protein
expressed to date could well have proven insurmountable to workers of
ordinary skill.
SUMMARY OF THE INVENTION
The potential artifacts and problems described above combine to suggest the
need for close scrutiny of any claims of successful cloning and expression
of human factor VIII. The success of the present invention is evidenced
by:
1) Immunological cross-reactivity of antibodies raised against
clone-derived factor VIII proteins with plasma-derived factor VIII
proteins.
2) Cross-reaction of neutralizing monoclonal antibodies raised against
human plasma factor VIII with protein encoded by the clone.
3) Identification of a genomic DNA corresponding to the factor VIII cDNA of
the invention as being located in the X-chromosome, where factor VIII gene
is known to be encoded.
4) Expression of a functional protein which exhibits:
a) Correction of factor VIII deficient plasma.
b) Activation of factor X to factor X.sub.a in the presence of factor
IX.sub.a, calcium and phospholipid.
c) Inactivation of the activity observed in a) and b) by antibodies
specific for factor VIII.
d) Binding of the activity to an immobilized monoclonal antibody column
specific for factor VIII.
e) Activation of the factor VIII activity by thrombin.
f) Binding of the activity to and subsequent elution from immobilized von
Willebrand factor.
Thus, the present invention is based upon the successful use of recombinant
DNA technology to produce functional human factor VIII, and in amounts
sufficient to prove identification and functionality and to initiate and
conduct animal and clinical testing as prerequisites to market approval.
The product, human factor VIII, is suitable for use, in all of its
functional forms, in the prophylactic or therapeutic treatment of human
beings diagnosed to be deficient in factor VIII coagulant activity.
Accordingly, the present invention, in one important aspect, is directed
to methods of diagnosing and treating classic hemophilia (or hemophilia A)
in human subjects using factor VIII and to suitable pharmaceutical
compositions therefor.
The present invention further comprises essentially pure, functional human
factor VIII. The product produced herein by genetically engineered
appropriate host systems provides human factor VIII in therapeutically
useful quantities and purities. In addition, the factor VIII hereof is
free of the contaminants with which it is ordinarily associated in its
non-recombinant cellular environment.
The present invention is also directed to DNA isolates as well as to DNA
expression vehicles containing gene sequences encoding human factor VIII
in expressible form, to transformant host cell cultures thereof, capable
of producing functional human factor VIII. In still further aspects, the
present invention is directed to various processes useful for preparing
said DNA isolates, DNA expression vehicles, host cell cultures, and
specific embodiments thereof. Still further, this invention is directed to
the preparation of fermentation cultures of said cell cultures.
Further, the present invention provides novel polypeptides comprising
moiety(ies) corresponding to functional segments of human factor VIII.
These novel polypeptides may represent the bioactive and/or antigenic
determinant segments of native factor VIII. For example, such polypeptides
are useful for treating hemophiliacs per se, and particularly those who
have developed neutralizing antibodies to factor VIII. In the latter
instance, treatment of such patients with polypeptides bearing the
requisite antigen determinant(s) could effectively bind such antibodies,
thereby increasing the efficiency of treatment with polypeptides bearing
the bioactive portions of human factor VIII.
The factor VIII DNA isolates produced according to the present invention,
encoding functional moiety(ies) of human factor VIII, find use in gene
therapy, restoring factor VIII activity in deficient subjects by
incorporation of such DNA, for example, via hematopoetic stem cells.
Particularly Preferred Embodiment
Human factor VIII is produced in functional form in a particularly suitable
host cell system. This system comprises baby hamster kidney cells (BHK-21
(C-13), ATCC No. CCL 10) which have been transfected with an expression
vector comprising DNA encoding human factor VIII, including 3'- and 5'-
untranslated DNA thereof and joined at the 3'- untranslated region with
3'- untranslated terminator DNA sequence, e.g., such as from hepatitis B
surface antigen gene. Expression of the gene is driven by transcriptional
and translational control elements contributed by the adenovirus major
late promoter together with its 5' spliced leader as well as elements
derived from the SV40 replication origin region including transcriptional
enhancer and promoter sequences. In addition, the expression vector may
also contain a DHFR gene driven by an SV40 early promoter which confers
gene amplification ability, and a selectable marker gene, e.g., neomycin
resistance (which may be provided via cotransfection with a separate
vector bearing neomycin resistance potential).
DESCRIPTION OF THE DRAWINGS
FIG. 1. Diagrammatic representation of the coagulation cascade (2).
FIG. 2. Melting of DNA in TMACl and 6.times. SSC. A: For each point ten
duplicate aliquots of .lambda. DNA were first bound to nitrocellulose
filters. These filters were then hybridized without formamide at
37.degree. C. as described in Methods. Pairs of spots were then washed in
6.times. SSC, 0.1 percent SDS (.quadrature.) or 3.0M TMACl, 50 mM Tris
HCl, pH 8.0, 0.1 percent SDS, 2 mM EDTA (0) in 2.degree. C. increments
from 38.degree. to 56.degree. C. The melting temperature is the point
where 50 percent of the hybridization intensity remained. B: A melting
experiment as in panel A was performed by binding aliquots of pBR322 DNA
to nitrocellulose filters. Probe fragments of various lengths were
generated by digestion of pBR322 with MspI, end-labeling of the fragments
with .sup.32 P, and isolation on polyacrylamide gels. The probe fragments
from 18 to 75 b were hybridized without formamide at 37.degree. C. and
those from 46 to 1374 b in 40 percent formamide at 37.degree. C. as
described in Methods. The filters were washed in 3.0M tetramethylammonium
chloride (TMACl), 50 mM Tris HCl, pH 8.0, 0.1 percent SDS, 2 mM EDTA in
3.degree. C. increments to determine the melting temperature. (0) melting
temperature determined for pBR322 MspI probe fragments, (.DELTA.) melting
temperatures in 3.0M TMACl from panel A for 11-17 b probes.
FIG. 3. Detection of the Factor VIII gene with probe 8.3. Left three
panels: Southern blots of 46,XY (1X, male) DNA and 49,XXXXY (4X) human DNA
digested with EcoRI and BamHI were hybridized in 6.times. SSC, 50 mM
sodium phosphate (pH 6.8), 5.times. Denhardt's solution, 0.1 g/l boiled,
sonicated salmon sperm DNA, 20 percent formamide at 42.degree. C. as
described in Methods. The three blots were washed in 1.times. SSC, 0.1
percent SDS at the temperature indicated. Lane 1, EcoRI 1X; lane 2, EcoRI
4X; lane 3, BamHI 1X; and lane 4, BamHI 4X. Lane M, end-labeled
.lambda.HindIII and .phi.X174 HaeIII digested marker fragments. Right
panel: One nitrocellulose filter from the .lambda./4X library screen
hybridized with probe 8.3. Arrows indicate two of the independent Factor
VIII positive clones. Hybridization and washing for the library screen was
as described above for the Southern blots, with a wash temperature of
37.degree. C.
FIG. 4. Map of the Human Factor VIII Gene. The top line shows the positions
and relative lengths of the 26 protein coding regions (Exons A to Z) in
the Factor VIII gene. The direction of transcription is from left to
right. The second line shows the scale of the map in kilobase pairs (kb).
The location of the recognition sites for the 10 restriction enzymes that
were used to map the Factor VIII gene are given in the next series of
lines. The open boxes represent the extent of human genomic DNA contained
in each of the .lambda. phage (.lambda.114, .lambda.120, .lambda.222,
.lambda.482, .lambda.599 and .lambda.605) and cosmid (p541, p542, p543,
p612, 613, p624) clones. The bottom line shows the locations of probes
used in the genomic screens and referred to in the text: 1)0.9 kb
EcoRI/BamHI fragment from p543; 2) 2.4 kb EcoRI/BamHI fragment from
.lambda.222; 3) 1.0 kb NdeI/BamHI triplet of fragments from .lambda.120;
4) oligonucleotide probe 8.3; 5) 2.5 kb StuI/EcoRI fragment from
.lambda.114; 6) 1.1 kb EcoRI/BamHI fragment from .lambda.482; 7) 1.1 kb
BamHI/EcoRI fragment from p542. Southern blot analysis of 46,XY and
49,XXXXY genomic DNA revealed no discernible differences in the
organization of the Factor VIII gene.
FIG. 5. Cosmid vector pGcos4. The 403 b annealed HincII fragment of
.lambda.c1857S7 (Bethesda Research Lab.) containing the cos site was
cloned in pBR322 from AvaI to PvuII to generate the plasmid pGcos1.
Separately, the 1624 b PvuII to NaeI fragment of pFR400 (49n), containing
an SV40 origin and promoter, a mutant dihydrofolate reductase gene, and
hepatitis B surface antigen termination sequences, was cloned into the
pBR322 AhaIII site to generate the plasmid mp33dhfr. A three-part ligation
and cloning was then performed with the 1497 b SphI to NdeI fragment of
pGcos1, the 3163 b NdeI to EcoRV fragment of mp33dhfr, and the 376 b EcoRV
to SphI fragment of pKT19 to generate the cosmid vector pGcos3. pKT19 is a
derivative of pBR322 in which the BamHI site in the tetracycline
resistance gene has the mutated nitroguanosine treatment. pGcos4 was
generated by cloning the synthetic 20mer, 5' AATTCGATCGGATCCGATCG, in the
EcoRI site of pGcos3.
FIG. 6. Map of pESVDA. The 342 b PvuII-HindIII fragment of SV40 virus
spanning the SV40 origin of replication and modified to be bounded by
EcoRI sites (73), the polyadenylation site of hepatitis B virus (HBV)
surface antigen (49n), contained on a 580 bp BamHI-BglII fragment, and the
pBR322 derivative pML (75) have been previously described. Between the
EcoRI site following the SV40 early promoter and the BamHI site of HBV was
inserted the PvuII-HindIII fragment (map coordinates 16.63-17.06 of
Adenovirus 2) containing the donor splice site of the first late leader
(position 16.65) immediately followed by the 840 bp HindIII-SacI fragment
of Adenovirus 2 (position 7.67-9.97) (49j), containing the Elb acceptor
splice site at map position 9.83. Between the donor and acceptor sites lie
unique BglII and HindIII sites for inserting genomic DNA fragments.
FIG. 7. Analysis of RNA transcripts from pESVDA vectors. Confluent 10 cm
dishes of COS-7 cells (77) were transfected with 2 .mu.g plasmid DNA using
the modified DEAE-dextran method (84) as described (73). RNA was prepared
4 days post-transfection from cytoplasmic extracts (49n) and
electrophoresed in denaturing formaldehyde-agarose gels. After transfer to
nitrocellulose, filters were hybridized with the appropriate .sup.32
P-labelled DNA as described in Methods. Filters were washed in 2.times.
SSC, 0.2 percent SDS at 42.degree. and exposed to Kodak XR5 film. The
position of the 28S and 18S ribosomal RNAs are indicated by arrow in each
panel.
The 9.4 kb BamHI fragment of .lambda.114 containing exon A (see FIG. 4) was
cloned into the BglII site of pESVDA (FIG. 6). Plasmid pESVDA111.6
contained the fragment inserted in the orientation such that the SV40
early promoter would transcribe the genomic fragment in the proper (i.e.,
sense) direction. pESVDA111.7 contains the 9.4 kb BamHI fragment in the
opposite orientation. Plasmid pESVDA.S127 contains the 12.7 kb SacI
fragment of .lambda.114 inserted (by blunt end ligation) into the BglII
site of pESVDA in the same orientation as pESVDA111.6.
A. Hybridization of filters containing total cytoplasmic RNA from cells
transfected with pESVDA, pESVDA111.7 and pESVDA111.6. pESVD RNA (lane 1),
pESVDA111.7 (lane 2), pESVDA111.6 (lanes 3-5). Probed with Factor 8 exon A
containing fragment (lanes 1-4) or 1800 b StuI/Bam fragment (lane 5).
Faint cross-hybridization is seen to 18S RNA.
B. Hybridization of RNA with StuI/BamHI probe ("intron robe"). RNA from: 1)
pESVDA, polyA.sup.- ; 2) pESVDA, polyA.sup.+ ; 3) pESVDA111.7, polyA.sup.-
; 4) pESVDA111.7, polyA.sup.+ ; 5) pESVDA111.6, polyA.sup.- ; 6)
pESVDA111.6, polyA.sup.+. The small dark hybridizing band seen in lanes
A5, B1, B3 and B5 probably represents hybridization to tRNA or to an Alu
repeat sequence found in this region.
C. Comparison of cytoplasmic RNA from pESVDA111.6 (lane 1) and pESVDA.S127
(lane 2) probed with exon A containing fragment. Note the slight size
increase in lane 2 representing additional exon sequences contained in the
larger genomic fragment.
FIG. 8. Sequence of pESVDA.S127 cDNA clone S36. The DNA sequence of the
human DNA insert is shown for the cDNA clone S36 obtained from the exon
expression plasmid pESVDA.S127 (see infra for details). Vertical lines
mark exon boundaries as determined by analysis of genomic and cDNA clones
of factor VIII, and exons are lettered as in FIG. 4. Selected restriction
endonuclease sites are indicated.
FIG. 9. cDNA cloning. Factor VIII mRNA is depicted on the third line with
the open bar representing the mature protein coding region; the hatched
area the signal peptide coding region, and adjacent lines the untranslated
regions of the message. The 5' end of the mRNA is at the left. Above this
line is shown the extent of the exon B region of the genomic clone
.lambda.222, and below the mRNA line are represented the six cDNA clones
from which were assembled the full length factor VIII clone (see text for
details). cDNA synthesis primers 1, 3, 4 and oligo(dT) are shown with
arrows depicting the direction of synthesis for which they primed.
Selected restriction endonuclease sites and a size scale in kilobases are
included.
FIG. 10 (A-C). Sequence of Human Factor VIII Gene. The complete nucleotide
sequence of the composite Factor VIII cDNA clone is shown with nucleotides
numbered at the left of each line. Number one represents the A of the
translation initiation codon ATG. Negative numbers refer to 5'
untranslated sequence. (mRNA mapping experiments suggest that Factor VIII
mRNA extends approximately 60 nucleotides farther 5' than position -109
shown here.) The predicted protein sequence is shown above the DNA.
Numbers above the amino acids are S1-19 for the predicted signal peptide,
and 1-2332 for the predicted mature protein. "Op" denotes the opal
translation stop codon TAG. The 3' polyadenylation signal AATAAA is
underlined and eight residues of the poly(A) tail (found in clone
.lambda.c10.3) are shown. The sequence homologous to the synthetic
oligonucleotide probe 8.3 has also been underlined (nucleotides
5557-5592). Selected restriction endonuclease cleavage sites are shown
above the appropriate sequence. Nucleotides 2671-3217 represent sequence
derived from genomic clones while the remainder represents cDNA sequence.
The complete DNA sequence of the protein coding region of the human factor
VIII gene was also determined from the genomic clones we have described.
Only two nucleotides differed from the sequence shown in this figure
derived from cDNA clones (except for nucleotides 2671-3217). Nucleotide
3780 (underlined) is G in the genomic clone, changing the amino acid codon
1241 from asp to glu. Nucleotide 8728 (underlined) in the 3' untranslated
region is A in the genomic clone.
FIG. 11. Assembly of full length recombinant factor VIII plasmid. See the
text section 8a for details of the assembly of the plasmid pSVEFVIII
containing the full length of human factor VIII cDNA. The numbering of
positions differs from those in the text and FIG. 10 by 72 bp.
FIG. 12. Assembly of the factor VIII expression plasmid. See the text
section 8b for details of the assembly of the plasmid pAML3p.8c1 which
directs the expression of functional human factor VIII in BHK cells.
FIG. 13. Western Blot analysis of factor VIII using fusion protein
antisera. Human factor VIII was separated on a 5-10 percent polyacrylamide
gradient SDS gel according to the procedure of (81). One lane of factor
VIII was stained with silver (80). The remaining lanes of factor VIII were
electrophoretically transferred to nitrocellulose for Western Blot
analysis. Radiolabeled standards were applied into lanes adjacent to
factor VIII in order to estimate the molecular weight of the observed
bands. As indicated, the nitrocellulose strips were incubated with the
appropriate antisera, washed, and probed with .sup.125 I protein A. The
nitrocellulose sheets were subjected to autoradiography.
FIG. 14. Analysis of fusion proteins using C8 monoclonal antibody. Fusion
proteins 1, 3 and 4 were analyzed by Western blotting analysis for
reactivity with the factor VIII specific monoclonal antibody C8.
FIG. 15. Elution profile for high pressure liquid chromatography (HPLC) of
factor VIII on a Toya Soda TSK 4000 SW column. The column was equilibrated
and developed at room temperature with 0.1 percent SDS in 0.1M sodium
phosphate, pH 7.0.
FIG. 16. Elution profile for reverse phase HPLC separation of factor VIII
tryptic peptides. The separation was performed on a Synchropak RP-P C-18
column (0.46 cm.times.25 cm, 10 microns) using a gradient elution of
acetonitrile (1 percent to 70 percent in 200 minutes) in 0.1 percent
trifluoroacetic acid. The arrow indicates the peak containing the peptide
with the sequence AWAYFSDVDLEK.
FIG. 17. Thrombin activation of purified factor VIII activity. The cell
supernatant was chromatographed on the C8 monoclonal resin, and dialyzed
to remove elution buffer. Thrombin (25ng) was added at time 0. Aliquots
were diluted 1:3 at the indicated times and assayed for coagulant
activity. Units per ml were calculated from a standard curve of normal
human plasma.
DETAILED DESCRIPTION
A. Definitions
As used herein, "human factor VIII" denotes a functional protein capable,
in vivo or in vitro, of correcting human factor VIII deficiencies,
characterized, for example, by hemophilia A. The protein and associated
activities are also referred to as factor VIIIC (FVIIIC) and factor VIII
coagulant antigen (FVIIICAg)(31a). Such factor VIII is produced by
recombinant cell culture systems active form(s) corresponding to factor
VIII activity native to human plasma. (One "unit" of human factor VIII
activity has been defined as that activity present in one milliliter of
normal human plasma.) The factor VIII protein produced herein is defined
by means of determined DNA gene and amino acid sequencing, by physical
characteristics and by biological activity.
Factor VIII has multiple degradation or processed forms in the natural
state. These are proteolytically derived from a precursor, one chain
protein, as demonstrated herein. The present invention provides such
single chain protein and also provides for the production per se or via in
vitro processing of a parent molecule of these various degradation
products, and administration of these various degradation products, which
have been shown also to be active. Such products contain functionally
active portion(s) corresponding to native material.
Allelic variations likely exist. These variations may be demonstrated by
one or more amino acid differences in the overall sequence or by
deletions, substitutions, insertions or inversions of one or more amino
acids in the overall sequence. In addition, the location of and degree of
glycosylation may depend on the nature of the host cellular environment.
Also, the potential exists, in the use of recombinant DNA technology, for
the preparation of various human factor VIII derivatives, variously
modified by resultant single or multiple amino acid deletions,
substitutions, insertions or inversions, for example, by means of site
directed mutagenesis of the underlying DNA. In addition, fragments of
human factor VIII, whether produced in vivo or in vitro, may possess
requisite useful activity, as discussed above. All such allelic
variations, glycosylated versions, modifications and fragments resulting
in derivatives of factor VIII are included within the scope of this
invention so long as they contain the functional segment of human factor
VIII and the essential, characteristic human factor VIII functional
activity remains unaffected in kind. Such functional variants or modified
derivatives are termed "human factor VIII derivatives" herein. Those
derivatives of factor VIII possessing the requisite functional activity
can readily be identified by straightforward in vitro tests described
herein. From the disclosure of the sequence of the human factor VIII DNA
herein and the amino acid sequence of human factor VIII, the fragments
that can be derived via restriction enzyme cutting of the DNA or
proteolytic or other degradation of human factor VIII protein will be
apparent to those skilled in the art.
Thus, human factor VIII in functional form, i.e., "functional human factor
VIII", is capable of catalyzing the conversion of factor X to Xa in the
presence of factor IXa, calcium, and phospholipid, as well as correcting
the coagulation defect in plasma derived from hemophilia A affected
individuals, and is further classified as "functional human factor VIII"
based on immunological properties demonstrating identity or substantial
identity with human plasma factor VIII.
"Essentially pure form" when used to describe the state of "human factor
VIII" produced by the invention means substantially free of protein or
other materials ordinarily associated with factor VIII when isolated from
non-recombinant sources, i.e. from its "native" plasma containing
environment.
"DHFR protein" refers to a protein which is capable of exhibiting the
activity associated with dihydrofolate reductase (DHFR) and which,
therefore, is required to be produced by cells which are capable of
survival on medium deficient in hypoxanthine, glycine, and thymidine (-HGT
medium). In general, cells lacking DHFR protein are incapable of growing
on this medium, and cells which contain DHFR protein are successful in
doing so.
"Expression vector" includes vectors which are capable of expressing DNA
sequences contained therein, where such sequences are operably linked to
other sequences capable of effecting their expression. These expression
vectors replicate in the host cell, either by means of an intact operable
origin of replication or by functional integration into the cell
chromosome. Again, "expression vector" is given a functional definition,
and any DLA sequence which is capable of effecting expression of a
specified DNA code disposed therein is included in this term as it is
applied to the specified sequence. In general, expression vectors of
utility in recombinant DNA techniques are often in the form of "plasmids"
which refer to circular double stranded DNA loops. However, the invention
is intended to include such other forms of expression vectors which serve
equivalent functions.
"DNA isolate" means the DNA sequence comprising the sequence encoding human
factor VIII, either itself or as incorporated into a cloning vector.
"Recombinant host cell" refers to cell/cells which have been transformed
with vectors constructed using recombinant DNA techniques. As defined
herein, factor VIII or functional segments thereof are produced in the
amounts achieved by virtue of this transformation, rather than in such
lesser amounts, and degrees of purity, as might be produced by an
untransformed, natural host source. Factor VIII produced by such
"recombinant host cells" can be referred to as "recombinant human factor
VIII".
Size units for DNA and RNA are often abbreviated as follows: b=base or base
pair; kb=kilo (one thousand) base or kilobase pair. For proteins we
abbreviate: D=Dalton; kD=kiloDalton. Temperatures are always given in
degrees Celsius.
B. Host Cell Cultures and Vectors
Useful recombinant human factor VIII may be produced, according to the
present invention, in a variety of recombinant host cells. A particularly
preferred system is described herein.
In general, prokaryotes are preferred for cloning of DNA sequences in
constructing the vectors useful in the invention. For example, E. coli K12
strain 294 (ATCC No. 31446) is particularly useful. Other microbial
strains which may be used include E. coli strains such as E. coli B, and
E. coli X1776 (ATTC No. 31537), and E. coli c600 and c600hfl, E. coli
W3110 (F.sup.-, .lambda..sup.-, prototrophic, ATTC No. 27325), bacilli
such as Bacillus subtilus, and other enterobacteriaceae such as Salmonella
typhimurium or Serratia marcesans, and various pseudomonas species. These
examples are, of course, intended to be illustrative rather than limiting.
In general, plasmid vectors containing replicon and control sequences which
are derived from species compatible with the host cell are used in
connection with these hosts. The vector ordinarily carries a replication
site, as well as marking sequences which are capable of providing
phenotypic selection in transformed cells. For example, E. coli is
typically transformed using pBR322, a plasmid derived from an E. coli
species (32). pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides easy means for identifying and selecting
transformed cells. The pBR322 plasmid, or other microbial plasmid, must
also contain, or be modified to contain, promoters which can be used by
the microbial organism for expression of its own proteins. Those promoters
most commonly used in recombinant DNA construction include the
.beta.-lactamase (penicillinase) and lactose promoter systems (33-35) and
a tryptophan (trp) promoter system (36, 37). While these are the most
commonly used, other microbial promoters have been discovered and
utilized, and details concerning their nucleotide sequences have been
published, enabling a skilled worker to ligate them functionally with
plasmid vectors (38).
In addition to prokaryotes, eukaryotic microbes, such as yeast cultures,
may also be used. Saccharomyces cerevisiae, or common baker's yeast, is
the most commonly used among eukaryotic microorganisms, although a number
of other strains are commonly available. For expression in Saccharomyces,
the plasmid YRp7, for example, (39-41) is commonly used. This plasmid
already contains the trp1 gene which provides a selection marker for a
mutant strain of yeast lacking the ability to grow in tryptophan, for
example, ATCC No. 44076 or PEP4-1 (42). The presence of the trp1 lesion as
a characteristic of the yeast host cell genome then provides an effective
environment for detecting transformation by growth in the absence of
tryptophan.
Suitable promoting sequences in yeast vectors include the promoters for
3-phosphoglycerate kinase (43) or other glycolytic enzymes (44, 45), such
as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,
phosphoglucose isomerase, and glucokinase. In constructing suitable
expression plasmids, the termination sequences associated with these genes
are also ligated into the expression vector 3' of the sequence desired to
be expressed to provide polyadenylation of the mRNA and termination. Other
promoters, which have the additional advantage of transcription controlled
by growth conditions, are the promoter regions for alcohol dehydrogenase
2, isocytochrome C, acid phosphatase, degradative enzymes associated with
nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate
dehydrogenase, and enzymes responsible for maltose and galactose
utilization. Any plasmid vector containing yeast-compatible promoter,
origin of replication and termination sequences is suitable.
Use of cultures of cells derived from multicellular organisms as cell hosts
is preferred, particularly for expression of underlying DNA to produce the
functional human factor VIII hereof, and reference is particularly had to
the preferred embodiment hereof. In principle, vertebrate cells are of
particular interest, such as VERO and HeLa cells, Chinese hamster ovary
(CHO) cell lines, and W138, BHK, COS-7 and MDCK cell lines. Expression
vectors for such cells ordinarily include (if necessary) (an) origin(s) of
replication, a promoter located in front of the gene to be expressed,
along with any necessary ribosome binding sites, RNA splice sites,
polyadenylation site, and transcriptional terminator sequences.
For use in mammalian cells, the control functions on the expression vectors
may be provided by viral material. For example, commonly used promoters
are derived from polyoma, Simian Virus 40 (SV40) and most particularly
Adenovirus 2. The early and late promoters of SV40 virus are useful as is
the major late promoter of adenovirus as described above. Further, it is
also possible, and often desirable, to utilize promoter or control
sequences normally associated with the desired gene sequence, provided
such control sequences are compatible with the host cell systems.
An origin of replication may be provided either by construction of the
vector to include an exogenous origin, such as may be derived from
adenovirus or other viral (e.g. Polyoma, SV40, VSV, BPV, etc.) source, or
may be provided by the host cell chromosomal replication mechanism, if the
vector is integrated into the host cell chromosome.
In selecting a preferred host cell for transfection by the vectors of the
invention which comprise DNA sequences encoding both factor VIII and DHFR
protein, it is appropriate to select the host according to the type of
DHFR protein employed. If wild type DHFR protein is employed, it is
preferable to select a host cell which is deficient in DHFR, thus
permitting the use of the DHFR coding sequence as a marker for successful
transfection in selective medium which lacks hypoxanthine, glycine, and
thymidine.
On the other hand, if DHFR protein with low binding affinity for MTX is
used as the controlling sequence, it is not necessary to use DHFR
resistant cells. Because the mutant DHFR is resistant to methotrexate, MTX
containing media can be used as a means of selection provided that the
host cells themselves are methotrexate sensitive. Most eukaryotic cells
which are capable of absorbing MTX appear to be methotrexate sensitive.
Alternatively, a wild type DHFR gene may be employed as an amplification
marker in a host cell which is not deficient in DHFR provided that a
second drug selectable marker is employed, such as neomycin resistance.
Examples which are set forth hereinbelow describe use of BHK cells as host
cells and expression vectors which include the adenovirus major late
promoter.
C. General Methods
If cells without formidable cell wall barriers are used as host cells,
transfection is carried out by the calcium phosphate precipitation method
(46). However, other methods for introducing DNA into cells such as by
nuclear injection or by protoplast fusion may also be used.
If prokaryotic cells or cells which contain substantial cell wall
constructions are used, the preferred method of transfection is calcium
treatment using calcium chloride (47).
Construction of suitable vectors containing the desired coding and control
sequences employs standard ligation techniques. Isolated plasmids or DNA
fragments are cleaved, tailored, and religated in the form desired to form
the plasmids required.
Cleavage is performed by treating with restriction enzyme (or enzymes) in
suitable buffer. In general, about 1 .mu.g plasmid or DNA fragments are
used with about 1 unit of enzyme in about 20 .mu.l of buffer solution for
1 hour. (Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Likewise, standard
conditions for use of T4 ligase, T4 polynucleotide kinase and bacterial
alkaline phosphatase are provided by the manufacturer.) After incubations,
protein is removed by extraction with phenol and chloroform, and the
nucleic acid is recovered from the aqueous fraction by precipitation with
ethanol. Standard laboratory procedures are available (48).
Sticky ended (overhanging) restriction enzyme fragments are rendered blunt
ended, for example, by either:
Fill in repair: 2-15 .mu.g of DNA were incubated in 50 mM NaCl , 10 mM Tris
(pH 7.5), 10 mM MgCl.sub.2, 1 mM dithiothreitol with 250 .mu.M each four
deoxynucleoside triphosphates and 8 units DNA polymerase Klenow fragment
at 24.degree. C. for 30 minutes. The reaction was terminated by phenol and
chloroform extraction and ethanol precipitation, or
S1 digestion: 2-15 .mu.g of DNA were incubated in 25 mM NaOAc (pH 4.5), 1
mM ZnCl.sub.2, 300 mM NaCl with 600 units S.sub.1 nuclease at 37.degree.
for 30 minutes, followed by phenol, chloroform and ethanol precipitation.
Synthetic DNA fragments were prepared by known phosphotriester (47a) or
phosphoramidite (47b) procedures. DNA is subject to electrophoresis in
agarose or polyacrylamide slab gels by standard procedures (48) and
fragments were purified from gels by electroelution (48a). DNA "Southern"
blot hybridization followed the (49a) procedure.
RNA "Northern" blot hybridizations followed electrophoresis in agarose slab
gels containing 6 percent formaldehyde. (48, 49b) Radiolabeled
hybridization probes are prepared by random calf thymus DNA primed
synthesis (49c) employing high specific activity .sup.32 P-labeled
nucleotide triphosphates (.sup.32 P: Amersham; Klenow DNA polymerase: BRL,
NEB or Boehringer-Mannheim). Short oligonucleotide probes may be
end-labelled with T4 polynucleotide kinase. "Standard salt" Southern
hybridization conditions ranged from: Hybridization in 5.times. SSC
(1.times. SSC=0.15M NaCl 0.015M Na.sub.3 citrate), 50 mM Na Phosphate pH
7, 10 percent dextran sulfate, 5.times. Denhardt's solution (1.times.
Denhardt's=0.02 percent ficoll, 0.02 percent polyvinylpyrrolidone, 0.02
percent bovine serum albumin), 20-100 .mu.g/ml denatured salmon sperm DNA,
0-50 percent formamide at temperatures ranging from 24.degree. to
42.degree. C., followed by washes in 0.2-1.times. SSC plus 0.1 percent SDS
at temperatures ranging from 24.degree.-65.degree. C. Dried filters were
exposed to Kodak XAR film using DuPont Lightning-Plus intensifying screens
at -80.degree. C. See, generally, (48).
For Northern blot screening of cell and tissue RNAs, hybridization was in
5.times. SSC, 5.times. Denhardt's solution, 10 percent dextran sulfate, 50
percent formamide, 0.1 percent SDS, 0.1 percent sodium pyrophosphate, 0.1
mg/ml E. coli tRNA at 42.degree. C. overnight with .sup.32 P-labeled probe
prepared from the 189 bp StuI/HincI fragment of .lambda.120 containing
exon A sequence. Wash conditions were 0.2.times. SSC, 0.1 percent SDS at
42.degree. C.
Human DNA was prepared from peripheral blood lymphocytes (46,XY) or
lymphoblast cells (49,XXXXY, N.I.G.M.S. Human Genetic Mutant Cell
Repository, Camden, N.J., No. GM1202A) (48). E. coli plasmid DNA was
prepared as in (48) and bacteriophage .lambda. DNA (48). Tissue RNA was
prepared by either the guanidinium thiocyanate method (48, 49f) or the
method of (49b). Polyadenylated RNA was isolated on oligo (dT) cellulose
(49h).
DNA sequence analysis was performed by the method of (49i).
For the .lambda./4X library, five 50 .mu.g aliquots of the 49, XXXXY DNA
was digested in a 1 ml volume with Sau3AI concentrations of 3.12, 1.56,
0.782, 0.39, and 0.195 U/ml for 1 hr at 37.degree. C. Test digestion and
gel analysis had shown that under these conditions at 0.782 U/ml Sau3AI,
the weight average size of the DNA was about 30 kb; thus these digests
generate a number average distribution centered at 15 kb. DNA from 5
digests was pooled, phenol and chloroform extracted, ethanol precipitated
and electrophoresed on a 6 g/l low-gelling temperature horizontal agarose
gel (48) (Seaplaque agarose, FMC Corporation), in two
5.6.times.0.6.times.0.15 cm slots. The 12-18 kb region of the gel was cut
out and the DNA purified by melting the gel slice as described in (48).
Charon 30 arms were prepared by digesting 50 .mu.g of the vector with BamHI
and isolating the annealed 31.9 kb arm fragment from a 6 g/l low-gelling
temperature agarose gel as described above. For construction of the
.lambda./4X library, the optimal concentration of Charon 30 BamHI arms and
12-18 kb Sau3A partial 49,XXXXY DNA was determined as described (48). The
ligated DNA was packaged with an in vitro extract, "packagene" (Promega
Biotec, Inc., Madison, Wis.). In a typical reaction about 1.3 .mu.g of
Charon 30 BamHI arms were ligated to 0.187 .mu.g of 12-18 kb Sau3A insert
DNA in a 10 .mu.l volume. Packaging the plating of the DNA gave about
1.3.times.10.sup.6 phage plaques. To generate the .lambda.4X library,
1.7.times.10.sup.6 phage were plated at 17000 phage per 150 cm plate.
These plates were grown overnight, scraped into 10 mM Tris HCl, pH 7.5,
0.1M NaCl, 10 mM MgCl.sub.2, 0.5 g/l gelatin, and centrifuged briefly, to
amplify the phage. Generally, a suitable number (0.5-2.times.10.sup.6) of
these phage were plated out and screened (48). In some cases the ligated
and in vitro packaged phage were screened directly without amplification.
For the isolation of .lambda.482, a clone containing a 22 kb BclI fragment
of the Factor VIII genome, and the BamHI arm fragments of the vector
.lambda.1059 (49) were isolated by gel electrophoresis. Separately, 100
.mu.g of DNA from the 49,XXXXY cell line was digested with BclI and the
20-24 kb fraction isolated by gel electrophoresis. About 0.8 .mu.g of
.lambda.1059 arms fragments and 5 percent of the isolated BclI DNA were
ligated in a volume of 10 .mu.l (48) to generate 712,000 plaques. Four
hundred thousand of those were screened in duplicate with 2.2 kb
StuI/EcoRI probe of .lambda.114.
The cosmid/4X library was generated from the 49,XXXXY DNA used to generate
the .lambda./4X library, except that great care was used in the DNA
isolation to avoid shearing or other breakage. The DNA was partially
cleaved with five concentrations of Sau3AI and the pooled DNA sized on a
100 to 400 g/l sucrose gradient (49). The fractions containing 35-45 kb
DNA were pooled, dialyzed, and ethanol precipitated. Arm fragments of the
cosmid vector pGcos4 were prepared following the principles described
elsewhere (50). In brief, two separate, equal aliquots of pGcos4 were cut
with SstI (an isoschizomer of SacI) or SalI and then treated with
bacterial alkaline phosphatase. These aliquots were then phenol and
chloroform extracted, pooled, ethanol precipitated and cut with BamHI.
From this digest two arm fragments of 4394 and 4002 b were isolated from a
low-gelling temperature agarose gel. These arm fragments were then ligated
to the isolated, 40 kb Sau3AI partial digest DNA. In a typical reaction,
0.7 .mu.g of pGcos4 arm fragments were ligated to 1 .mu.g of 40 kb human
4X DNA in a volume of 10 .mu.l (48). This reaction was then packaged in
vitro and used to infect E. coli HB101, a recA.sup.31 strain (48). This
reaction generated about 120,000 colonies when plated on tetracycline
containing plates. About 150,000 cosmids were screened on 20 150-mm plates
in duplicate as described, with overnight amplification on
chloramphenicol-containing plates (48).
Double-stranded cDNA was prepared as previously described (36, 67)
employing either oligo(dT)12-18 or synthetic deoxyoligonucleotide 16-mers
as primers for first-strand synthesis by reverse transcriptase. Following
isolation by polyacrylamide gels, cDNA of the appropriate size (usually
600 bp or greater) was either C-tailed with terminal transferase, annealed
together with G-tailed PstI-digested pBR322 and transformed into E. coli
strain DH1 (76), or ligated with a 100-fold molar excess of synthetic DNA
EcoRI adaptors, reisolated on a polyacrylamide gel, inserted by ligation
in EcoRI-digested .lambda.GT10, packaged into phage particles and
propagated on E. coli strain C600hfl (68). As a modification of existing
procedures an adaptor consisting of a complementary synthetic DNA 18-mer
and 22-mer (5'-CCTTGACCGTAAGACATG and 5'AATTCATGTCTTACGGTCAAGG) was
phosphorylated at the blunt terminus but not at the EcoRI cohesive
terminus to permit efficient ligation of the adaptor to double-stranded
cDNA in the absence of extensive self-ligation at the EcoRI site. This
effectively substituted for the more laborious procedure of ligating
self-complementary EcoRI linkers to EcoRI methylase-treated
double-stranded cDNA, and subsequently removing excess linker oligomers
from the cDNA termini by EcoRI digestion. To improve the efficiency of
obtaining cDNA clones >3500 bp extending from the poly(A) to the nearest
existing 3' factor VIII probe sequences made available by genomic cloning
(i.e., exon A), second-strand cDNA synthesis was specifically primed by
including in the reaction a synthetic DNA 16-mer corresponding to a
sequence within exon B on the mRNA sense strand.
D. Adenovirus Subcloning
Adenovirus 2 DNA was purchased from Bethesda Research Laboratories (BRL).
The viral DNA was cleaved with HindIII and electrophoresed through a 5
percent polyacrylamide gel (TBE buffer). The region of the gel containing
the HindIII B fragment (49j) was excised and the DNA electroeluted from
the gel. After phenol-chloroform extraction, the DNA was concentrated by
ethanol precipitation and cloned into HindIII-cleaved pUC13 (49k) to
generate the plasmid pAdHindB. This HindIII subclone was digested with
HindIII and SalI, and a fragment was isolated spanning adenoviral
coordinates 17.1-25.9 (49j). This fragment was cloned into HindIII, SalI
cleaved pUC13 to generate the plasmid pUCHS. From pAdHindB the SalI to
XhoI fragment, coordinates 25.9-26.5, was isolated and cloned into pUCHS
at the unique SalI site to create pUCHSX. This plasmid reconstructs the
adenoviral sequences from position 17.1 within the first late leader
intervening sequence to the XhoI site at position 26.5 within the third
late leader exon.
The adenovirus major late promoter was cloned by excising the HindIII C, D,
and E fragments (which comigrate) from the acrylamide gel, cloning them
into pUC13 at the HindIII site, and screening for recombinants containing
the HindIII C fragment by restriction analysis. This subclone was digested
with SacI, which cleaved at position 15.4, 5' of the major late promoter
(49j) as well as within the polylinker of pUC13. The DNA was
recircularized to form pMLP2, containing the SacI to HindIII fragment
(positions 15.4-17.1) cloned in the SacI and HindIII sites of pUC13.
E. Construction of Neomycin Resistance Vector
The neomycin resistance marker contained within E. coli transposon 5 was
isolated from a Tn5 containing plasmid (49l). The sequence of the neomycin
resistance gene has been previously published (49m). The neo fragment was
digested with BglII, which cleaves at a point 36 bp 5' of the
translational initiation codon of the neomycin phosphotransferase gene,
and treated with exonuclease Bal31. The phosphotransferase gene was
excised with BamHI, which cleaves the DNA 342 bp following the
translational termination codon, and inserted into pBR322 between a
filled-in HindIII site and the BamHI site. One clone, pNeoBal6, had the
translational initiation codon situated 3 bp 3' of the filled in HindIII
site (TCATCGATAAGCTCGCATG . . . ). This plasmid was digested with ClaI and
BamHI, whereupon the 1145 bp fragment spanning the phosphotransferase gene
was isolated and inserted into the mammalian expression vector pCVSVEHBS
(see infra.). The resultant plasmid, pSVENeoBa16, situates the neomycin
phosphotransferase gene 3' of the SV40 early promoter and 5' of the
polyadenylation site of the HBV surface antigen gene (49n). When
introduced into mammalian tissue culture cells, this plasmid is capable of
expressing the phosphotransferase gene and conferring resistance to the
aminoglycoside G418 (49o).
F. Transfection of Tissue Culture Cells
The BHK-21 cells (ATCC) are vertebrate cells grown in tissue culture. These
cells, as is known in the art, can be maintained as permanent cell lines
prepared by successive serial transfers from isolated normal cells. These
cell lines are maintained either on a solid support in liquid medium, or
by growth in suspensions containing support nutrients.
The cells are transfected with 5 .mu.g of desired vector (4 .mu.g
pAML3P.8cl and 1 .mu.g pSVEneoBal6) as prepared above using the method of
(49p).
The method insures the interaction of a collection of plasmids with a
particular host cell, thereby increasing the probability that if one
plasmid is absorbed by a cell, additional plasmids would be absorbed as
well (49q). Accordingly, it is practicable to introduce both the primary
and secondary coding sequences using separate vectors for each, as well as
by using a single vector containing both sequences.
G. Growth of Transfected Cells and Expression of Peptides
The BHK cells which were subjected to transfection as set forth above were
first grown for two days in non-selective medium, then the cells were
transferred into medium containing G418 (400 .mu.g/ml), thus selecting for
cells which are able to express the plasmid phosphotransferase. After 7-10
days in the presence of the G418, colonies became visible to the naked
eye. Trypsinization of the several hundred colonies and replating allowed
the rapid growth of a confluent 10 cm dish of G418 resistant cells.
This cell population consists of cells representing a variety of initial
integrants. In order to obtain cells which possessed the greatest number
of copies of the FVIII expression plasmid, the cells were next incubated
with an inhibitor of the DHFR protein.
H. Treatment with Methotrexate
The G418 resistant cells are inhibited by methotrexate (MTX), a specific
inhibitor of DHFR at concentrations greater than 50 nN. Consistent with
previous studies on the effects of MTX on tissue culture cells, cells
resistant to MTX by virtue of expression of the multiple copies of the
DHFR gene contained within the FVIII expression vector are selected for,
and a concomitant increase in expression of the FVIII encoding sequences
can be observed. By stepwise increasing the amount of MTX, amplification
of the plasmid pAML3P.8cl is affected, thus increasing the copy number.
The upper limit of the amplification is dependent upon many factors,
however cells resistant to millimolar concentrations of MTX possessing
hundreds or thousands of copies of the DHFR expression (and thus the FVIII
expression) plasmid may be selected in this manner.
For Factor VIII expression, G418-resistant BHK cells which arose after
transfection with pAML3P.8cl and pSVENeoBa16 were incubated with media
containing 100 nM and 250 nM NTX as described (49r). After 7-10 days,
cells resistant to 250 nM MTX were assayed for Factor VIII expression by
activity, radioimmunoassay and mRNA Northern analysis.
I. Factor VIII antibodies
A variety of polyclonal and monoclonal antibodies to Factor VIII were used
throughout this work. CC is a polyclonal antibody derived from the plasma
of a severely affected hemophiliac (49s). C8 is a neutralizing monoclonal
antibody which binds to the 210 kD portion of Factor VIII (49t). C10 is a
monoclonal antibody with properties similar to C8 and was isolated
essentially as described by (49t). A commercial neutralizing monoclonal
antibody which binds the 80 kD portion of Factor VIII was obtained from
Synbiotic Corp., San Diego, Calif., Product No. 10004. C7F7 is a
neutralizing monoclonal antibody that binds to the 80 kD portion of Factor
VIII. C7F7 was induced and purified as follows: Six-week-old female BALB/c
mice were multiply inoculated with approximately 10 .mu.g of purified
Factor VIII and splenocytes fused with X63-Ag8.653 mouse myeloma cells
(49u) three days after the final inoculation. The hybridization procedure
and isolation of hybrid cells by cloning methods followed previously
described protocols (49r). Specific antibody producing clones were
detected by solid phase RIA procedures (49w). Positive clones were
subsequently assayed for coagulation prolongation capacity by APTT assay
described above. Monoclonal C7F7 was expanded by growth in syngeneic
animals; antibody was purified from ascites fluids by protein A-Sepharose
CL-4B chromatography (49x).
J. Radioimmune Assays for Factor VIII
Two radioimmune assays (RIA) were developed to assay Factor VIII produced
from BHK and other cell lines. Both are two stage assays in which the CC
antibody bound to a solid support is used to bind Factor VIII (49t). This
immune complex is then detected with I.sup.125 labeled C10 antibody (210
kD specific) or I.sup.125 labeled C7F7 antibody (80 kD specific).
Briefly, the two-stage RIAs are performed as follows: the 96 wells of a
microtiter dish are coated overnight with 100 .mu.l of 50 mM NaHCO.sub.3
buffer, pH 9.6 containing 2.5 mg/l of CC antibody which has been purified
by protein A-sepharose chromatography (49x). The wells are washed three
times with 200 .mu.l of PBS containing 0.05 percent Tween 20 and blocked
with 200 .mu.l of PBS containing 0.1 percent gelatin and 0.01 percent
methiolate for 1 to 2 hours. The wells are washed as before and 100 .mu.l
of sample added and incubated overnight. The wells are washed and 100
.mu.l of I.sup.125 labeled (82) C10 or C7F7 antibody (1000 cpm/.mu.l )
added and incubated 6 to 8 hours. The wells are washed again and counted.
The standard curve is derived from samples of normal plasma diluted 1:10
to 1:320.
K. Factor VIII Monoclonal Antibody Column
A human factor VIII monoclonal antibody column was prepared by incubation
of 1.0 mg of C8 antibody (in 0.1M NaHCO.sub.3, pH8.5) with 1.0 ml of
Affi-Gel 10 (Bio-Rad Laboratories, Richmond, Calif.) for four hours at
4.degree. C. Greater than 95 percent of the antibody was coupled to the
gel, as determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories).
The gel was washed with 50 volumes of water and 10 volumes of 0.5M
imidazole, pH6.9, containing 0.15M NaCl.
L. Chromatography of Media on Monoclonal Column
Media was applied to the monoclonal antibody column (1 ml of resin) and
washed with 0.05M imidazole buffer, pH6.4, containing 0.15M NaCl until
material absorbing at 280 nm was washed off. The column was eluted with
0.05M imidazole, pH6.4, containing 1.0M KI and 20 percent ethylene glycol.
Samples were diluted for assay and dialized for subsequent analysis.
M. Preparation of Factor VIII Fusion Proteins and Fusion Protein Antisera
E. coli containing the plasmids constructed for fusion protein expression
were grown in M-9 media at 37.degree. C. Fusion protein expression was
induced by the addition of indole acrylic acid at a final concentration of
50 .mu.g/mL for time periods of 2.5 to 4 hours. The cells were harvested
by centrifugation and frozen until use.
The cell pellets for fusion 3 were suspended in 100 mL of 20 mM sodium
phosphate, pH 7.2, containing 10 .mu.g/mL lysozyme and 1 .mu.g/mL each of
RNase and DNase. The suspension was stirred for 30 minutes at room
temperature to thoroughly disperse the cell pellet. The suspension was
then sonicated for four minutes (pulsed at 60 percent power). The solution
was centrifuged at 8000 rpm in a Sorvall RC-2B centrifuge in a GSA rotor.
The pellet was resuspended n 100 mL of 0.02M sodium phosphate, pH 7.2. The
suspension was layered over 300 mL of 60 percent glycerol. The sample was
centrifuged at 4000 rpm for 20 minutes in an RC-3B centrifuge. Two layers
resulted in the glycerol. Both pellet and the bottom glycerol layer showed
a single protein band of the expected molecular weight of 25,000 daltons
when analyzed on SDS polyacrylamide gels. The pellet was dissolved in
0.02M sodium phosphate buffer containing 0.1 percent SDS. The resuspended
pellet and the lower glycerol layer were dialyzed against 0.02M ammonium
bicarbonate, pH 8.0, to remove glycerol. The solution was lyophilized and
redissolved in 0.01M sodium phosphate buffer containing 0.1 percent SDS,
and frozen until use.
The cell pellets for fusion proteins 1 and 4 were suspended in 0.05M Tris,
pH 7.2, containing 0.3M sodium chloride and 5 mM EDTA. Lysozyme was added
to a concentration of 10 .mu.g/mL. Samples were incubated for 5 minutes at
room temperature. NP-40 was added to 0.2 percent and the suspension
incubated in ice for 30 minutes. Sodium chloride was added to yield a
final concentration of 3M and DNase added (1 .mu.g/mL). The suspension was
incubated 5 minutes at room temperature. The sample was centrifuged and
the supernatant discarded. The pellet was resuspended in a small volume of
water and recentrifuged. The cell pellets were dissolved in solutions
containing 0.1 percent to 1 percent SDS and purified by either preparative
SDS polyacrylamide gel electrophoresis followed by electroelution of the
fusion protein band, or by HPLC on a TSK 3000 column equilibrated with
0.1M sodium phosphate containing 0.1 percent SDS.
Rabbit antisera was produced by injecting New Zealand white rabbits with a
sample of fusion protein suspended in Freund's complete adjuvant (first
injection) following by boosts at two week intervals using the sample
suspended in Freund's incomplete adjuvant. After six weeks, sera was
obtained and analyzed by Western Blot analysis for reactivity with human
plasma derived factor VIII proteins.
N. Assays for Detection of Expression of Factor VIII Activity
Correction of Hemophilia A plasma--Theory--Factor VIII activity is defined
as that activity which will correct the coagulation defect of factor VIII
deficient plasma. One unit of factor VIII activity has been defined as
that activity present in one milliliter of normal human plasma. The assay
is based on observing the time required for formation of a visible fibrin
clot in plasma derived from a patient diagnosed as suffering from
hemophilia A (classic hemophilia). In this assay, the shorter the time
required for clot formation, the greater the factor VIII activity in the
sample being tested. This type of assay is referred to as activated
partial thromboplastin time (APTT). Commercial reagents are available for
such determinations (for example, General Diagnostics Platelin Plus
Activator; product number 35503).
Procedure--All coagulation assays were conducted in 10.times.75 mm
borosilicate glass test tubes. Siliconization was performed using SurfaSil
(product of Pierce Chemical Company, Rockford, Ill.) which had been
diluted 1 to 10 with petroleum ether. The test tubes were filled with this
solution, incubated 15 seconds, and the solution removed. The tubes were
washed three times with tap water and three times with distilled water.
Platelin Plus Activator (General Diagnostics, Morris Plains, N.J.) was
dissolved in 2.5 ml of distilled water according to the directions on the
packet. To prepare the sample for coagulation assays, the Platelin plus
Activator solution was incubated at 37.degree. C. for 10 minutes and
stored on ice until use. To a siliconized test tube was added 50
microliters of Platelin plus Activator and 50 microliters of factor VIII
deficient plasma (George King Biomedical Inc, Overland Park, Kans.). This
solution was incubated at 37.degree. C. for a total of nine minutes. Just
prior to the end of the nine minute incubation of the above solution, the
sample to be tested was diluted into 0.05M Tris-HCl, pH 7.3, containing
0.02 percent bovine serum albumin. To the plasma/activator suspension was
added 50 microliters of the diluted sample, and, at exactly nine minutes
into the incubation of the suspension, the coagulation cascade was
initiated by the addition of 50 microliters of calcium chloride (0.033M).
The reaction mixture was quickly mixed and, with gentle agitation of the
test tube, the time required for the formation of a visible fibrin clot to
form was monitored. A standard curve of factor VIII activity can be
obtained by diluting normal plasma (George King Biomedical, Inc., Overland
Park, Kans.) 1:10, 1:20, 1:50, 1:100, and 1:200. The clotting time is
plotted versus plasma dilution on semilog graph paper. This can then be
used to convert a clotting time into units of factor VIII activity.
O. Chromogenic Peptide Determination
Theory--Factor VIII functions in the activation of factor X to factor
X.sub.a in the presence of facton IX.sub.a, phospholipid, and calcium
ions. A highly specific assay has been designed wherein factor IX.sub.a,
factor X, phospholipid, and calcium ions are supplied. The generation of
factor X.sub.a in this assay is therefore dependent upon the addition of a
source of factor VIII activity. The more factor VIII added to the assay,
the more factor X.sub.a is generated. After allowing the generation of
factor X.sub.a, a chromogenic peptide substrate is added to the reaction
mixture. This peptide is specifically cleaved by factor X.sub.a, is not
effected by factor X, and is only slowly cleaved by other proteases.
Cleavage of the peptide substrate releases a para-nitro-anilide group
which has absorbance at 405 nm, while the uncleaved peptide substrate has
little or no absorbance at this wavelength. The generation of absorbance
due to cleavage of the chromogenic substrate is dependent upon the amount
of factor X.sub.a in the test mixture after the incubation period, the
amount of which is in turn dependent upon the amount of functional factor
VIII in the test sample added to the reaction mixture. This assay is
extremely specific for factor VIII activity and should be less subject to
potential false positives when compared to factor VIII deficient plasma
assay.
Procedure--Coatest factor VIII was purchased from Helena Laboratories,
Beaumont, Tex. (Cat. No. 5293). The basic procedure used was essentially
that provided by the manufacturer for the "End Point Method" for samples
containing less than 5 percent factor VIII. Where indicated, the times of
incubation were prolonged in order to make the assay more sensitive. For
certain assays the volumes of reagents recommended by the manufacturer
were altered. This change in the protocol does not interfere with the
overall results of the assay.
The chromogenic substrate (S-2222+I-2581) for factor X.sub.a was dissolved
in 10 milliliters of water, resulting in a substrate concentration of 2.7
millimoles per liter. This substrate solution was aliquoted and stored
frozen at -20.degree. C. The FIX.sub.a +FX reagent contained the factor
IX.sub.a and factor X and was dissolved in 10 milliliters of water. The
solution was aliquoted and stored frozen at -70.degree. C. until use. Also
supplied with the kit were the following solutions: 0.025 molar calcium
chloride; phospholipid (porcine brain); and Buffer Stock Solution (diluted
one part of Stock Solution to nine parts of water for the assay, resulting
in a final concentration of 0.06M Tris-HCl, pH 7.3, containing 0.02
percent bovine albumin). These solutions were stored at 4.degree. C. until
use.
The phospholipid +FIX.sub.a +FX reagent is prepared by mixing one volume of
phospholipid with five volumes of FIX.sub.a +FX reagent.
The following procedure was employed:
______________________________________
Sample Reagent
Reagent Tube Blank
______________________________________
Phospholipid + FIX.sub.a + FX
200 .mu.l 200 .mu.l
Test sample 100 --
Buffer working solution
-- 100
Mix well and incubate at 37.degree. C. for 4 minutes
100 100
Calcium chloride
Mix well and incubate at 37.degree. C. exactly 10
200 200
minutes S-2222 + I-2581
Mix well and incubate at 37.degree. C. exactly 10
100 100
minutes Acetic acid (50 percent)
Mix well
______________________________________
The absorbance of the sample at 405 nm was determined against the reagent
blank in a spectrophotometer within 30 minutes.
The absorbance at 405 was related to factor VIII units by calibrating the
assay using a standard normal human plasma (George King Biomedical,
Overland Park, Kans.).
EXAMPLE OF PREFERRED EMBODIMENT
1. General Strategy for Obtaining the Factor VIII Gene
The most common process of obtaining a recombinant DNA gene product is to
screen libraries of cDNA clones obtained from mRNA of the appropriate
tissue or cell type. Several factors contributed to use also of an
alternative method of screening genomic DNA for the factor VIII gene.
First, the site of synthesis of factor VIII was unknown. Although the
liver is frequently considered the most likely source of synthesis, the
evidence is ambiguous. Synthesis in liver and possibly spleen have been
suggested by organ perfusion and transplantation studies (56). However,
factor VIII activity is often increased in patients with severe liver
failure (56a). Recent conflicting studies employing monoclonal antibody
binding to cells detect highest levels of the protein in either liver
sinusoidal endothelial (51), hepatocyte (52) or lymph node cells (followed
in amount by lung, liver and spleen; (53)). In contrast, the factor VIII
related antigen (von Willebrand Factor) is almost certainly synthesized by
endothelial cells (54). Not only is the tissue source uncertain, the
quantity of factor VIII in plasma is extremely low. The circulating
concentration of about 100-200 ng/ml (55) is about 1/2,000,000 the molar
concentration of serum albumin, for example. Thus, it was not clear that
cDNA libraries made from RNA of a given tissue would yield factor VIII
clones.
Based on these considerations, it was decided to first screen recombinant
libraries of the human genome in bacteriophage lambda (henceforth referred
to as genomic libraries). Although genomic libraries should contain the
factor VIII gene, the likely presence of introns might present obstacles
to the ultimate expression of the recombinant protein. The general
strategy was to:
1. Identify a genomic clone corresponding to a sequenced portion of the
human factor VIII protein.
2. Conduct a "genomic walk" to obtain overlapping genomic clones that would
include the entire mRNA coding region.
3. Use fragments of the genomic clones to identify by hybridization to RNA
blots tissue or cell sources of factor VIII mRNA and then proceed to
obtain cDNA clones from such cells.
4. In parallel with no. 3, to express portions of genomic clones in SV40
recombinant "exon expression" plasmids. RNA transcribed from these
plasmids after transfection of tissue culture (cos) cells should be
spliced in vivo and would be an alternative source of cDNA clones suitable
for recombinant factor VIII protein expression.
The actual progress of this endeavor involved simultaneous interplay of
information derived from cDNA clones, genomic clones of several types, and
SV40 recombinant "exon expression" clones, which, of necessity, are
described separately below.
2. Genomic Library Screening Procedures
The factor VIII gene is known to reside on the human X chromosome (56). To
increase the proportion of positive clones, genomic libraries were
constructed from DNA obtained from an individual containing 4X
chromosomes. (The lymphoblast cell line is karyotyped 49,XXXXY; libraries
constructed from this DNA are referred to herein as "4X libraries").
49,XXXXY DNA was partially digested with Sau3AI and appropriate size
fractions were ligated into .lambda. phage or cosmid vectors. Details of
the construction of these .lambda./4X and cosmid/4X libraries are given
below. The expected frequency of the factor VIII gene in the .lambda./4X
library is about one in 110,000 clones and in the cosmid library about one
in 40,000.
These libraries were screened for the factor VIII gene with synthetic
oligonucleotide probes based on portions of the factor VIII protein
sequence. These oligonucleotide probes fall into two types, a single
sequence of 30 to 100 nucleotides based on codon choice usage analysis
(long probes) and a pool of probes 14-20 nucleotides long specifying all
possible degeneracy combinations for each codon choice (short probes).
The main advantage of long probes is that they can be synthesized based on
any 10-30 amino acid sequence of the protein. No special regions of low
codon redundancy need be found. Another advantage is that since an exact
match with the gene sequence is not necessary (only stretches of
complementarity of 10-14 nucleotides are required), interruption of
complementarity due to presence of an intron, or caused by gene
polymorphism or protein sequencing error, would not necessarily prevent
usable hybridization. The disadvantage of long probes is that only one
codon is selected for each amino acid. We have based our choice of codons
on a table of mammalian codon frequency (57), and when this gave no clear
preference, on the codon usage of the Factor IX gene (58). Since the
expected sequence match of the long probes is unknown, the hybridization
stringency must be determined empirically for each probe. This was
performed by hybridization to genomic DNA blots and washes at various
stringencies.
The advantage of short probes is that every codon possible is synthesized
as a pool of oligonucleotides. Thus if the amino acid sequence is correct,
a short probe should always hybridize to the gene of interest. The main
limitation is the complexity of the pool of sequences that can be
synthesized. Operationally a pool of 32 different sequences might be
considered as a maximum pool size given the signal to noise limitations of
hybridization to genomic libraries. This means that only protein sequences
in regions of low codon redundancy can be used. A typical probe would be a
pool of 16 17-mers specifying all possible sequences over a 6 amino acid
fragment of protein sequence.
As with long probes, the hybridization stringency used for short probes had
been determined empirically. This is because under ordinarily used
hybridization conditions (6.times. SSC), the stability of the hybrids
depends on the two factors--the length and the G-C content; stringent
conditions for the low G-C content probes are not at all stringent for the
high G-C content ones. A typical pool of 16 17-mers might have a range of
41 to 65 percent G-C and these probes will melt in 6.times. SSC over a
10.degree. C. temperature range (from 48.degree.-58.degree. C.). Since the
correct sequence within the pool of 16 is not known in advance, one uses a
hybridization stringency just below 48.degree. C. to allow hybridization
of the lowest G-C content sequence. However, when screening a large number
of clones, this will give many false positives of shorter length and
higher G-C content. Since the change in melting temperature is 1.degree.
to 2.degree. C. per base pair match, probe sequences as short as 12 or 13
of the 17 will also bind if they have a high G-C content. At random in the
human genome a pooled probe of 16 17-mers will hybridize with 1200 times
as many 13 base sequences as 17 base sequences.
A hybridization technique was developed for short probes which equalizes
the stability of G-C and A-T base pairs and greatly enhances the utility
of using short probes to screen libraries of high DNA sequence complexity.
In FIG. 2A is plotted the melting temperature of 4 short probes under
ordinary (6.times. SSC) and 3.0M TMACl wash conditions. In 3.0M TMACl the
probes melt as a nearly linear function of length, while in 6.times. SSC,
the melting is greatly influenced by the G-C content. The high melting
temperature in 6.times. SSC of the 13-mer that is 65 percent G-C clearly
demonstrates this conclusion. FIG. 2B shows the melting temperature in
3.0M TMACl as a function of length for 11 to thousands of bases. This
figure allows the rapid selection of hybridization conditions for a probe
with an exact match of any length desired.
The TMACl hybridization procedure has great utility whenever an exact
sequence match of some known length is desired. Examples of this technique
include: 1. Screening of a human genomic library with a pool of 16
17-mers. We have used a 3.0M TMACl wash at 50.degree. C., which allows
hybridization of only 17, 16, and a few 15 base sequences. The large
number of high G-C content probes of lower homology are thus excluded. 2.
If a short probe screen yields too many positives to sequence easily, the
mostly likely candidates can be found by a TMACl melting procedure.
Replicas of the positives are hybridized and washed at 2.degree. C.
intervals (for 17-mers (which melt at 54.degree. C.) 46.degree.,
48.degree., 50.degree., 52.degree., 54.degree., and 56.degree. C. would be
used). The positives that melt at the highest temperature will match the
probe most closely. With a standard of known sequence the homology can be
predicted .+-.1 base or better for a 17-mer. 3. Similarly, if a long probe
screen yields too many positives, pooled short probes based on the same
protein sequence can be synthesized. Since one member of this pool would
contain a perfect match, TMACl melting experiments could refine the choice
of best candidate positives. 4. In site directed mutagenesis, an
oligonucleotide typically 20 long with 1 or more changes in the center is
synthesized. The TMACl wash procedure can easily distinguish the parental
and mutant derivatives even for a 1 base mismatch in the middle of a
20-mer. This is because the desired mutation matches the probe exactly.
The wash conditions can simply be determined from FIG. 2B. 5. Selection of
one particular gene out of a family of closely related genes. A melting
experiment similar to that described above has been used to select one
particular gene out of a collection of 100 very similar sequences.
3. First Isolation of the Factor VIII Genomic Clone
Factor VIII enriched preparations were prepared from human cryoprecipitate
by polyelectrolyte chromatography and immunoadsorption as previously
described (79). This material was dialyzed into 0.1 percent sodium dodecyl
sulfate (SDS) and 1 percent ammonium bicarbonate, lyophilized, and stored
at -20.degree. C. until use.
Due to contamination of the factor VIII preparations by other plasma
proteins, further fractionation was required in order to purify the factor
VIII as well as separate the various polypeptide chains believed to arise
from the factor VIII. This was accomplished by chromatography of the
protein on Toya Soda TSK 4000 SW columns using high pressure liquid
chromatography in the presence of SDS. Such chromatography separates the
proteins by molecular size.
The lyophilized protein was reconstituted in distilled water and made 1
percent SDS and 0.1M sodium phosphate, pH 7.5. The TSK column
(0.75.times.50 cm; Alltech, Deerfield, Ill.) was equilibrated at room
temperature with 0.1 percent SDS in 0.1M sodium phosphate, pH 7.0. Samples
of approximately 0.15 to 0.25 mL were injected and the column was
developed isocratically at a flow rate of 0.5 mL per minute. The
absorbance was monitored at 280 nm and fractions of 0.2 mL were collected.
A representative elution profile is shown in FIG. 15. Aliquots were
analyzed by sodium dodecyl sulfate gel electrophoresis on gradient gels of
5 percent to 10 percent polyacrylamide and analyzed by silver staining
(80). The material which eluted after 25 minutes corresponded to a doublet
of proteins at 80,000 and 78,000 D. The fractions containing these
proteins were pooled as indicated by bar in FIG. 15, from three separate
preparative TSK runs, and stored at -20 degrees until use.
The purified 80,000 dalton protein from the TSK fractionation (0.8 nmoles)
was dialyzed overnight against 8M urea, 0.36M Tris-HCl, pH 8.6, and 3.3 mM
ethylenediamine-tetraacetic acid under a nitrogen atmosphere. Disulfide
bonds were reduced by the inclusion of 10 mM dithiothreitol in the above
dialysis buffer. The final volume was 1.5 ml. The cysteines were alkylated
with 15 microliters of 5M iodoacetic acid (dissolved in 1M NaOH). The
reaction was allowed to proceed for 35 minutes at room temperature in the
dark, and the alkylation reaction was quenched by the addition of
dithiothreitol to a final concentration of 100 mM. The protein solution
was dialyzed against 8M urea in 0.1M ammonium bicarbonate for four hours.
The dialysis solution was changed to gradually dilute the urea
concentration (8M, 4M, 2M, 1M, and finally 0.5M urea) over a period of 24
hours. Tryptic digestion was performed on the reduced, alkylated 80,000
dalton protein by the addition of TPCK-treated trypsin (Sigma Chem. Co.)
at a weight ratio of 1 part trypsin to 30 parts factor VIII protein. The
digestion was allowed to continue for 12 hours at 37.degree. C. The
reaction mixture was frozen until use. HPLC separation of the tryptic
peptides was performed on a high resolution Synchropak RP-P C-18 column
(0.46.times.25 cm, 10 microns) at room temperature with a Spectra-Physics
8000 chromatograph. Samples of approximately 0.8 mL were injected and the
column was developed with a gradient of acetonitrile (1 percent to 70
percent in 200 minutes) in 0.1 percent trifluoroacetic acid. The
absorbance was monitored at 210 nm and 280 nm (FIG. 16). Each peak was
collected and stored at 4.degree. C. until subjected to sequence analysis
in a Beckman spinning cup sequencer with on-line PTH amino acid
identification. The arrow in FIG. 16, eluting at approximately 23 percent
acetonitrile, indicates the peak containing the peptide with the sequence
AWAYFSDVDLEK. This sequence was used to generate the oligonucleotide probe
8.3 for human genomic library screening.
Long and short probes were synthesized based on the considerations just
discussed. The second long probe used was based on the sequence of a 12
amino acid factor VIII tryptic fragment, AWAYFSDVDLEK. The DNA sequence
chosen to synthesize for this probe was
5'-CTTTTCCAGGTCAACGTCGGAGAAATAAGCCCAAGC. This probe (called 8.3) was first
tested in genomic blot hybridizations. FIG. 3A shows genomic Southern
blots of normal male (1X) and 49,XXXXY (4X) DNA hybridized with labeled
8.3 probe and washed at various stringencies. Even at the highest
stringency (1.times. SSC, 46.degree. C.) a single band of 3.8 kb (EcoRI)
and 9.4 kb (BamHI) was observed. The intensity of this band had a ratio of
about 1:4 in the 1X and 4X lanes as would be expected for the X-linked
factor VIII gene. Control experiments had demonstrated that a known
X-linked gene probe (Factor IX) gave the expected 1:4 hybridization ratio,
while an autosomal gene (albumin) gave a 1:1 ratio.
Based on these genomic blot results, the 8.3 probe was used to screen the
.lambda./4X library. 500,000 phage were grown on fifty 150 mm plates and
duplicate nitrocellulose filters were hybridized with .sup.32 P-labeled
8.3 probe at a wash stringency of 1.times. SSC, 37.degree. C. (FIG. 3).
Upon retesting, 15 strongly hybridizing and 15 more weakly hybridizing
clones were obtained. DNA was prepared from these isolated plaques,
cleaved with restriction endonucleases, and blot hybridized with probe
8.3. Many of the strongly hybridizing clones yielded a hybridizing EcoRI
fragment of 3.8 kb, the same size detected in the genomic blot. In
addition, all strongly hybridizing clones displayed an identical 262 base
pair Sau3AI fragment upon hybridization with the 8.3 probe. Sau3AI
fragments were cloned into the single-stranded phage vector M13mp8 (86),
screened by hybridization, and sequenced by the dideoxy procedure. The DNA
sequence of the 262 bp fragment showed considerable homology with the 8.3
probe. The homology included regions of continuous matches of 14 and 10 bp
with an overall homology of 83 percent. The first ten residues of the
peptide fragment agreed with that deduced from the DNA sequence of the
recombinant clones and they were preceded by a lysine codon as expected
for the product of a tryptic digest. The final two predicted residues did
not match the DNA sequence. However, the DNA at this juncture contained a
good consensus RNA splice donor sequence (60, 61) followed shortly by stop
codons in all three possible reading frames. This suggested the presence
of an intron beginning at this position. (This suggestion was confirmed
with cDNA clones described below.) An open reading frame extended almost
400 b 5' of the region of homology. In this region several consensus
splice acceptor sequences were identified. Inspection of the DNA-predicted
protein sequence for this region revealed matches with protein sequence of
several additional tryptic peptide fragments of factor VIII. This
demonstrated that an exon of a genomic clone for human factor VIII had
been obtained.
4. Extension of Genomic Clones: .lambda.Library Genome Walking
Initially 8 independent factor VIII genomic clones were obtained from the
.lambda./4X library. These contained overlapping segments of the human
genome spanning about 28 kb. From the estimated size of the factor VIII
protein, it was assumed that the complete gene would encompass 100-200 kb,
depending on the length of introns. Hence the collection of overlapping
clones was expanded by "genome walking".
The first step in this process was the mapping of restriction endonuclease
cleavage sites in the existing genomic clones (FIG. 4). DNA from the
clones was digested with restriction enzymes singly or in combinations,
and characterized by gel electrophoresis (followed by Southern blot
hybridization in some cases). DNA fragments generated by EcoRI and BamHI
digestion were subcloned into pUC plasmid vectors (59) for convenience.
Restriction mapping, DNA sequence analysis, and blot hybridizations with
the 8.3 probe determined the gene orientation.
Next, single copy fragments near the ends of the 28 kb region were
identified as "walk" probes. Digests of cloned DNA were blot hybridized
with total .sup.32 P-labeled human DNA. With this technique only fragments
containing sequences repeated more than about 50 times in the genome will
hybridize (87, 88). Non-hybridizing candidate walk probe fragments were
retested for repeated sequences by hybridization to 50,000 phage from the
.lambda./4X library.
In the 5' direction, a triplet of 1 kb probe fragments was isolated from
.lambda.120 DNA digested with NdeI and BamHI (see FIG. 4). One million
.lambda./4X bacteriophage were screened with this probe. A resulting
clone, .lambda.222, was shown to extend about 13 kb 5' of .lambda.120 (see
FIG. 4).
In the 3' direction, a 2.5 kb StuI/EcoRI restriction fragment of
.lambda.114 was identified as a single copy walk probe. Exhaustive
screening of the .lambda./4X, and subsequently other .lambda./human
genomic libraries, failed to yield extending clones. Under-representation
of genomic regions in .lambda. libraries has been observed before (62). It
was decided to specifically enrich genomic DNA for the desired sequences
and construct from it a limited bacteriophage library.
Southern blot hybridization of human genomic DNA with the 2.5 kb StuI/EcoRI
probe showed a 22 kb hybridizing BclI restriction fragment. Restriction
mapping showed that cloning and recovery of this fragment would result in
a large 3' extension of genomic clones. Human 49,XXXXY DNA was digested
with BclI, and a size fraction of about 22 kb was purified by gel
electrophoresis. This DNA was ligated into the BamHI site of the
bacteriophage vector .lambda.1059 and a library was prepared. (The
previously used vector, Charon 30, could not accommodate such a large
insert.) Six hybridizing clones were obtained from 400,000 phage screened
from this enriched library. The desired clone, designated .lambda.482,
extended 17 kb further 3' than our original set of overlapping genomic
clones (FIG. 4).
5. Genome Walking: Cosmid Clones
A new genomic library was constructed with cosmid vectors. Cosmids (63), a
plasmid and bacteriophage hybrid, can accommodate approximately 45 kb of
insert, about a three-fold increase over the average insert size of the
.lambda./4X DNA library. A newly constructed cosmid vector, pGcos4, has
the following desirable attributes:
1. A derivative of the tetracycline resistance gene of pBR322 was used that
did not contain a BamHI site. This allowed a BamHI site to be put
elsewhere in the plasmid and to be used as the cloning site. Tetracycline
resistance is somewhat easier to work with than the more commonly used
ampicillin resistance due to the greater stability of the drug. 2. The 403
b HincII fragment of .lambda. containing the cos site was substituted for
the 641 b AvaI/PvuII fragment of pBR322 so that the copy number of the
plasmid would be increased and to remove pBR322 sequences which interfere
with the transformation of eukaryotic cells (75). 3. A mutant
dihydrofolate reductase gene with an SV40 origin of replication and
promoter was included in the pGcos4 vector. In this way any fragments
cloned in this vector could then be propagated in a wide range of
eucaryotic cells. It was expected this might prove useful in expressing
large fragments of genomic DNA with their natural promoters. 4. For the
cloning site, a synthetic 20-mer with the restriction sites EcoRI, PvuI,
BamHI, PvuI, and EcoRI was cloned into the EcoRI site from pBR322. The
unique BamHI site is used to clone 35-45 b Sau3A1 fragments of genomic
DNA. The flanking EcoRI sites can be used for subcloning the EcoRI
fragments of the insert. The PvuI sites can be used to cut out the entire
insert in most cases. PvuI sites are exceedingly rare in eucaryotic DNA
and are expected to occur only once every 134,000 b based on dinucleotide
frequencies of human DNA.
FIG. 5 gives the scheme for constructing the cosmid vector, pGcos4. 35-45
kb Sau3A1 fragments of 49, XXXXY DNA were cloned in this vector. About
150,000 recombinants were screened in duplicate with a 5' 2.4 kb
EcoRI/BamHI fragment of 222 and a 3' 1 kb EcoRI/BamHI fragment of
.lambda.482 which were single copy probes identified near the ends of the
existing genomic region. Four positive cosmid clones were isolated and
mapped. FIG. 4 includes cosmids p541, p542 and p543. From this screen,
these cosmid clones extended the factor VIII genomic region to a total of
114 kbp. Subsequent probing with cDNA clones identified numerous exons in
the existing set of overlapping genomic clone, but indicated that the
genomic walk was not yet complete. Additional steps were taken in either
direction.
A 3' walk probe was prepared from a 1.1 kb BamHI/EcoRI fragment of p542
(FIG. 4). This probe detected the overlapping cosmid clone p613 extending
about 35 kb farther 3'. At a later time, the full Factor VIII message
sequence was obtained by cDNA cloning (see below). When a 1.9 kb EcoRI
cDNA fragment containing the 3'-terminal portion of the cDNA was
hybridized to Southern blots of human genomic and cosmid cloned DNA, it
identified a single 4.9 kb EcoRI band and 5.7, 3.2 and 0.2 kb BamHI bands
in both noncloned (genomic) and p613 DNA. This implied that the 3' end of
the gene had now been reached, as we later confirmed by DNA sequence
analysis.
A 5' walk probe was prepared from a 0.9 kb EcoRI/pamHI fragment of p543. It
detected an overlapping cosmid clone p612, which slightly extended the
overlapping region. The 5'-most genomic clones were finally obtained by
screening cosmid/4X and .lambda./4X libraries with cDNA derived probes. As
shown in FIG. 4, .lambda.1599, .lambda.605 and p624 complete the set of
recombinant clones spanning Factor VIII gene. (These clones overlap and
contain all of the DNA of this region of the human genome with the
exception of an 8.4 kb gap between p624 and .lambda.599 consisting solely
of intron DNA.) Together, the gene spans 200 kb of the human X chromosome.
This is by far the largest gene yet reported. Roughly 95 percent of the
gene is comprised of introns which must be properly processed to produce
template mRNA for the synthesis of Factor VIII protein.
The isolation of the factor VIII gene region in .lambda. and cosmid
recombinant clones is not sufficient to produce a useful product, the
factor VIII protein. Several approaches were followed to identify and
characterize the protein coding (exon) portions of the gene in order to
ultimately construct a recombinant expression plasmid capable of directing
the synthesis of active factor VIII protein in transfected microorganisms
or tissue culture cells. Two strategies failed to yield substantially
useful results: further screening of genomic clones with new
oligonucleotide probes based on protein sequencing, and the use of
selected fragments of genomic clones as probes to RNA blot hybridizations.
However, coding regions for the factor VIII protein were isolated with the
use of SV40 "exon expression" vectors, and, ultimately, by cDNA cloning.
6. SV40 exon expression vectors
It is highly unlikely that a genomic region of several hundred kb could be
completely characterized by DNA sequence analysis or directly used to
synthesize useful amounts of factor VIII protein. Roughly 95 percent of
the human factor VIII gene comprises introns (intervening sequences) which
must be removed artificially or by eukaryotic RNA splicing machinery
before the protein could be expressed. A procedure was created to remove
introns from incompletely characterized restriction fragments of genomic
clones using what we call SV40 expression vectors. The general concept
entails inserting fragments of genomic DNA into plasmids containing an
SV40 promoter and producing significant amounts of recombinant RNA which
would be processed in the transfected monkey cos cells. The resulting
spliced RNA can be analyzed directly or provide material for cDNA cloning.
In theory at least, this technique could be used to assemble an entire
spliced version of the factor VIII gene.
Our first exon expression constructions used existing SV40 cDNA vectors
that expressed the hepatitis surface antigen gene (73). However, the
genomic factor VIII fragments cloned into these vectors gave no observable
factor VIII RNA when analyzed by blot hybridization. It was surmised that
the difficulty might be that in the course of these constructions the exon
regions of the cDNA vectors had been joined to intron regions of the
factor VIII gene. To circumvent these difficulties, the exon expression
vector pESVDA (ATCC Accession No. 99,776, deposited with American Type
Culture Collection (ATCC, 12301 Parklawn Drive, Rockville, Md. 20852,
U.S.A. under the Budapest Treaty on Oct. 28, 1996) was constructed as
shown in FIG. 6. This vector contains the SV40 early promoter, the
Adenovirus II major late first splice donor site, intron sequences into
which the genomic factor VIII fragments could be cloned, followed by the
Adenovirus II E1b splice acceptor site and the hepatitis B surface antigen
3' untranslated and polyadenylation sequences (49j).
Initially the 9.4 kb BamHI fragment and the 12.7 kb SstI fragment of
.lambda.114 were cloned in the intron region of pESVDA (see FIG. 6).
Northern blot analysis of the RNA synthesized by these two constructions
after transfection of cos cells is shown in FIG. 7. With the 9.4 kb BamHI
construction, a hybridizing RNA band of about 1.8 kb is found with probes
for exon A, and hepatitis 3' untranslated sequence. To examine the RNA for
any new factor VIII exons, a 2.0 kbp StuI/BamHI fragment of .lambda.114,
3' of exon A, was hybridized in a parallel lane. This probe also showed an
RNA band of 1.8 kb demonstrating the presence of additional new factor
VIII exons in this region. Each of these three probes also hybridized to
an RNA band from a construction containing the 12.7 kb SstI genomic
fragment. This RNA band was about 2.1 kb. This observation suggested that
an additional 200-300 bp of exon sequences were contained in this
construction 3' of the BamHI site bordering the 9.4 kb BamHI fragment.
Control experiments showed that this system is capable of correctly
splicing known exon regions. A 3.2 kb genomic HindlII fragment of murine
dhfr spanning exons III and IV was cloned in pESVDA. An RNA band of 1 kb
was found with a murine dhfr probe. This is the size expected if the exons
are spliced correctly. Constructions with the 9.4 kb BamHI factor VIII or
3.2 kb dhfr genomic fragments in the opposite orientation, gave no
observable RNA bands with any of the probes (FIG. 7).
A cDNA copy of the RNA from the 12.7 kb SstI construction was cloned in
pBR322 and screened. One nearly full length (1700 bp) cDNA clone (S36) was
found. The sequence of the 950 bp SstI fragment containing all of the
factor VIII insert and a portion of the pESVDA vector on either side is
presented in FIG. 8. The sequence begins and ends with the Adenovirus
splice donor and acceptor sequences as expected. In between there are 888
bp of factor VIII sequence including exon A. The 154 bp preceding and the
568 bp following exon A contain several factor VIII 80K tryptic fragments,
confirming that these are newly identified exons. Sequences of the genomic
region corresponding to these exons showed that the 154 bp 5' of exon A
are contained in one exon, C, and that the region 3' of exon A is composed
of 3 exons, D, E, and I of 229, 183 and 156 bp, respectively. Each of
these exons is bounded by a reasonable splice donor and acceptor site (60,
61).
Subsequent comparison of the S36 exon expression cDNA with the factor VIII
cell line cDNA clones showed that all the spliced factor VIII sequence in
S36 is from factor VIII exons. This included as expected exons C, A, D, E,
and I. However, 47 bp of exon A were missing at the C, A junction and
exons F, G, and H had been skipped entirely. The reading frame shifts
resulting from such aberrant RNA processing showed that it could not
correspond exactly to the factor VIII sequence. At the C, A junction a
good consensus splice site was utilized rather than the authentic one. The
different splicing of the S36 clone compared with the authentic factor
VIII transcript may be because only a portion of the RNA primary
transcript was expressed in the cos cell construction. Alternatively, cell
type or species variability may account for this difference.
7. cDNA Cloning
a. Identification of a cell line producing Factor VIII mRNA
To identify a source of RNA for the isolation of factor VIII. cDNA clones,
polyadenylated RNA was isolated from numerous human cell lines and tissues
and screened by Northern blot hybridization with the 189 bp StuI-HincII
fragment from the exon A region of .lambda.120. Poly(A).sup.+ RNA from the
CH-2 human T-cell hybridoma exhibited a hybridizing RNA species. The size
of the hybridizing RNA was estimated to be about 10 kb. This is the size
mRNA expected to code for a protein of about 300 kD. By comparison with
control DNA dot-blot hybridizations (66), the amount of this RNA was
determined to be 0.0001-0.001 percent of the total cellular poly(A)+ RNA
in the CH-2 cell line. This result indicated that isolation of factor VIII
cDNA sequences from this source would require further enrichment of
specific sequences or otherwise entail the screening of extremely large
numbers of cDNA clones.
b. Specifically Primed cDNA Clones
The DNA sequence analysis of Factor VIII genomic clones allowed the
synthesis of 16 base synthetic oligonucleotides to specifically prime
first strand synthesis of cDNA. Normally, oligo(dT) is used to prime cDNA
synthesis at the poly(A) tails of mRNA. Specific priming has two
advantages over oligo(dT). First, it serves to enrich the cDNA clone
population for factor VIII. Second, it positions the cDNA clones in
regions of the gene for which we possessed hybridization probes. This is
especially important in cloning such a large gene. As cDNA clones are
rarely longer than 1000-2000 base pairs, oligo(dT) primed clones would
usually be undetectable with a probe prepared from most regions of the
factor VIII gene. The strategy employed was to use DNA fragments and
sequence information from the initial exon A region to obtain specifically
primed cDNA clones. We proceeded by obtaining a set of overlapping cDNA
clones in the 5' direction based upon the characterization of the earlier
generation of cDNA clones. In order to derive the more 3' region of cDNA,
we employed cDNA and genomic clone fragments from 3' exons to detect
oligo(dT) primed cDNA clones. Several types of cDNA cloning procedures
were used in the course of this endeavor and will be described below.
The initial specific cDNA primer, 5'-CAGGTCAACATCAGAG ("primer 1"; see FIG.
9) was synthesized as the reverse complement of the 16 3'-terminal
residues of the exon A sequence. C-tailed cDNA was synthesized from 5
.mu.g of CH-2 cell poly(A)+ RNA with primer 1, and annealed into G-tailed
pBR322 as described generally in (67). Approximately 100,000 resulting E.
coli transformants were plated on 100 150 mm dishes and screened by
hybridization (48) with the 189 bp StuI/HincII fragment from the exon A
region of the genomic clone .lambda.120 (FIG. 4). One bona fide
hybridizing clone ("p1.11") was recovered (see FIG. 9). DNA sequence
analysis of p1.11 demonstrated identity with our factor VIII genomic
clones. The 447 bp cDNA insert in p1.11 contained the first 104 b of
genomic exon A (second strand synthesis apparently did not extend back to
the primer) and continued further into what we would later show to be
exons B and C. The 5' point of divergence with exon A sequence was
bordered by a typical RNA splice acceptor site (61).
Although the feasibility of obtaining factor VIII cDNA clones from the CH-2
cell line had now been demonstrated, further refinements were made.
Efforts of several types were made to further enrich CH-2 RNA for factor
VIII message. A successful strategy was to combine specifically primed
first strand cDNA synthesis with hybrid selection of the resulting single
stranded cDNA. Primer 1 was used with 200 .mu.g of poly(A).sup.+ CH-2 RNA
to synthesize single stranded cDNA. Instead of using DNA polymerase to
immediately convert this to double stranded DNA, the single stranded DNA
was hybridized to 2 .mu.g of 189 bp StuI/HincII genomic fragment DNA which
had been immobilized on activated ABM cellulose paper (Schleicher and
Schuell "Transa-Bind"; see (48). Although RNA is usually subject to hybrid
selection, the procedure was applied after cDNA synthesis in order to
avoid additional manipulation of the rare, large and relatively labile
factor VIII RNA molecules. After elution, the material was converted to
double stranded cDNA, size selected, and 0.5 ng of recovered DNA was
C-tailed and cloned into pBR322 as before. Approximately 12,000
recombinant clones were obtained and screened by hybridization with a 364
bp Su3A/StuI fragment derived from the previous cDNA clone p1.11. The
probe fragment was chosen deliberately not to overlap with the DNA used
for hybrid selection. Thus avoided was the identification of spurious
recombinants containing some of the StuI/HincII DNA fragment which is
invariably released from the DBM cellulose. 29 hybridizing colonies were
obtained. This represents a roughly 250-fold enrichment of desired clones
over the previous procedure.
Each of the 29 new recombinants was characterized by restriction mapping
and the two longest (p3.12 and p3.48; FIG. 9) were sequenced. These cDNA
clones extended about 1500 bp farther 5' than p1.11. Concurrent mapping
and sequence analysis of cDNA and genomic clones revealed the presence of
an unusually large exon (exon B, FIG. 4) which encompassed p3.12 and
p3.48. Based on this observation, DNA sequence analysis of the genomic
clone .lambda.222 was extended to define the extent of this exon. Exon B
region contained an open reading frame of about 3 kb. 16 mer primers 2 and
3 were synthesized to match sequence within this large exon in the hope of
obtaining a considerable extension in cDNA cloning.
At this point, it was demonstrated that a bacteriophage based cDNA cloning
system could be employed, enabling production and screening of vast
numbers of cDNA clones without prior enrichment by hybrid selection.
.lambda.GT10 (68) is a phage .lambda. derivative with a single EcoRI
restriction site in its repressor gene. If double stranded cDNA fragments
are flanked by EcoRI sites they can be ligated into this unique site.
Insertion of foreign DNA into this site renders the phage repressor minus,
forming a clear plaque. .lambda.GT10 without insert forms turbid plaques
which are thus distinguishable from recombinants. In addition to the great
transformation efficiency inherent in phage packaging, .lambda. cDNA
plaques are more convenient to screen at high density than are bacterial
colonies.
Double stranded cDNA was prepared as before using primer 3,
5'-AACTCTGTTGCTGCAG (located about 550 bp downstream from the postulated
5' end of exon B). EcoRI "adaptors" were ligated to the blunt ended cDNA.
The adaptors consisted of a complementary synthetic 18 mer and 22 mer of
sequence 5'-CCTTGACCGTAAGACATG and 5'-AATTCATGTCTTACGGTCAAGG. The 5' end
of the 18mer was phosphorylated, while the 5' end of the 22mer retained
the 5'-OH with which it was synthesized. Thus, when annealed and ligated
with the cDNA, the adaptors form overhanging EcoRI sites which cannot
self-ligate. This allows one to avoid EcoRI methylation of cDNA and
subsequent EcoRI digestion which follows linker ligation in other
published procedures (83). After gel isolation to size select the cDNA and
remove unreacted adapters, an equimolar amount of this cDNA was ligated
into EcoRI cut .lambda.GT10, packaged and plated on E. coli c600hfl. About
3,000,000 clones from 1 .mu.g of poly(A).sup.+ RNA were plated on 50 150
mm petri dishes and hybridization screened with a 300 bp HinfI fragment
from the 5' end of exon B. 46 duplicate positives were identified and
analyzed by EcoRI digestion. Several cDNA inserts appeared to extend about
2500 bp 5' of primer 3. These long clones were analyzed by DNA sequencing.
The sequences of the 5' ends of .lambda.13.2 and .lambda.13.27 are shown
in FIG. 10. They possessed several features which indicated that we had
reached the 5' end of the coding region for factor VIII. The initial 109
bp contained stop codons in all possible reading frames. Then appeared an
ATG triplet followed by an open reading frame for the rest of the 2724 bp
of the cDNA insert in .lambda.13.2. Translation of the sequence following
the initiator ATG gives a 19 amino acid sequence typical of a secreted
protein "leader" or "pre" sequence (69). Its salient features are two
charged residues bordering a 10 amino acid hydrophobic core. Following
this putative leader sequence is a region corresponding to amino terminal
residues obtained from protein sequence analysis of 210 kD and 95 kD
thrombin digest species of factor VIII.
c. Oligo(dT) primed cDNA clones
Several thousand more 3' bases of factor VIII mRNA remained to be converted
into cDNA. The choice was to prime reverse transcription with oligo(dT)
and search for cDNA clones containing the 3' poly(A).sup.+ tails of mRNA.
However, in an effort to enrich the clones and to increase the efficiency
of second strand DNA synthesis, established procedures were replaced with
employment of a specific primer of second strand cDNA synthesis. The
16-mer primer 4, 5'-TATTGCTGCAGTGGAG, was synthesized to represent message
sense sequence at a PstI site about 400 bp upstream of the 3' end of exon
A (FIG. 9). mRNA was reverse transcribed with oligo(dT) priming, primer 4
was added with DNA polymerase for second strand synthesis, and EcoRI
adapted cDNA then ligated into .lambda.GT10 as before. 3,000,000 plaques
were screened with a 419 bp PstI/HincII fragment contained on p3.12, lying
downstream from primer 4. DNA was prepared from the four clones recovered.
These were digested, mapped, and blot hybridized with further downstream
genomic fragments which had just been identified as exons using SV40 exon
expression plasmids described above. Three of the four recombinants
hybridized. The longest, .lambda.10.44, was approximately 1,800 base
pairs. The DNA sequence of .lambda.10.44 showed that indeed second strand
synthesis began at primer 4. It contained all exon sequences found in the
SV40 exon expression clone S36 and more. However, the open reading frame
of .lambda.10.44 continued to the end of the cDNA. No 3' untranslated
region nor poly(A) tail was found. Presumably second strand synthesis had
not gone to completion.
To find clones containing the complete 3' end, we rescreened the same
filters with labeled DNA from .lambda.10.44. 24 additional clones were
recovered and mapped, and the two longest (.lambda.10.3 and
.lambda.10.9.2) were sequenced. They contained essentially identical
sequences which overlapped .lambda.10.44 and added about 1900 more 3' base
pairs. 51 base pairs beyond the end of the .lambda.10.44 terminus, the DNA
sequence showed as TGA translation stop codon followed by an apparent 3'
untranslated region of 1805 base pairs. Diagnostic features of this region
are stop codons dispersed in all three reading frames and a poly(A) signal
sequence, AATAAA 189), followed 15 bases downstream with a poly(A) stretch
at the end of the cDNA (clone .lambda.10.3 contains 8 A's followed by the
EcoRI adapter at this point, while .lambda.10.9.2 contains over 100 A's at
its 3' end).
d. Complete cDNA Sequence
The complete sequence of overlapping clones is presented in FIG. 10. It
consists of a continuous open reading frame coding for 2351 amino acids.
Assuming a putative terminal signal peptide of 19 amino acids, the
"mature" protein would therefore have 2332 amino acids. The calculated
molecular weight for this protein is about 267,000 daltons. Taking into
account possible glycosylation, this approximates the molecular weight of
native protein as determined by SDS polyacrylamide gel electrophoresis.
The "complete" cDNA length of about 9000 base pairs (depending on the
length of 3' poly(A)) agrees with the estimated length of the mRNA
determined by Northern blot hybridization. The 5' (amino terminal coding)
region contains substantial correspondence to the peptide sequence of 210
kD derived factor VIII material and the 3' (carboxy terminal coding)
region contains substantial correspondence to the peptide sequence of 80
kD protein.
8. Expression of Recombinant Factor VIII
a. Assembly of full length clone
In order to express recombinant Factor VIII, the full 7 kb protein coding
region was assembled from several separate cDNA and genomic clones. We
describe below and in FIG. 11 the construction of three intermediate
plasmids containing the 5', middle, and 3' regions of the gene. The
intermediates are combined in an expression plasmid following an SV40
early promoter. This plasmid in turn serves as the starting point for
various constructions with modified terminal sequences and different
promoters and selectable markers for transformation of a number of
mammalian cell types.
The 5' coding region was assembled in a pBR322 derivative in such a way as
to place a ClaI restriction site before the ATG start codon of the Factor
VIII signal sequence. Since no other ClaI site is found in the gene, it
becomes a convenient site for refinements of the expression plasmid. The
convenient ClaI and SacI containing plasmid pT24-10 (67a) was cleaved with
HindlII, filled in with DNA polymerase, and cut with SacI. A 77 b
AluI/SacI was recovered from the 5' region of the Factor VIII cDNA clone
.lambda.13.2 and ligated into this vector to produce the intermediate
called pF8Cla-Sac. (The AluI site is located in the 5' untranslated region
of Factor VIII and the SacI site 10 b beyond the initiator ATG at
nucleotide position 10 in FIG. 10; the nucleotide position of all
restriction sites to follow will be numbered as in FIG. 10 beginning with
the A of the initiator codon ATG.) An 85 b ClaI/SacI fragment containing
11 bp of adaptor sequence (the adaptor sequence 5' ATCGATAAGCT is entirely
derived from pBR322) was isolated from pF8Cla-Sac and ligated along with
an 1801 b SacI/KpnI (nucleotide 1811) fragment from .lambda.13.2 into a
ClaI/KpnI vector prepared from a pBR322 subclone containing a HindIII
fragment (nuc. 1019-2277) of Factor VIII. This intermediate, called
pF8Cla-Kpn, contained the initial 2277 coding nucleotides of Factor VIII
preceded by 65 5' untranslated base pairs and the 11 base pair ClaI
adapter sequence. pF8Cla-Kpn was opened with KpnI and SphI (in the pBR322
portion) to serve as the vector fragment in a ligation with a 466 b
KpnI/HindIII fragment derived from an EcoRI subclone of .lambda.13.2 and a
1654 b HindIII/SphI (nuc. 4003) fragment derived from the exon B
containing subclone p222.8. This produced pF8Cla-Sph containing the first
3931 b of Factor VIII coding sequence.
The middle part of the coding region was derived from a three-piece
ligation combining fragments of three pBR322/cDNA clones or subclones.
p3.48 was opened with BamHI (nuc. 4743) and SalI (in pBR322 tet region) to
serve as vector. Into these sites were ligated a 778 b BamHI/NdeI (nuc.
5520) fragment from p3.12 and a 2106 b NdeI/SalI (in pBR322) fragment from
the subclone p.lambda.10.44R1.9. Proper ligation resulted in a
tetracycline resistant plasmid pF8Sca-RI.
The most 3' portion of Factor VIII cDNA was cloned directly into an SV40
expression vector. The plasmid pCVSVEHBV contains an SV40 early promoter
followed by a polylinker and the gene for the Hepatitis B surface antigen.
›pCVSVEHBV, also referred to as pCVSVEHBS, is a slight variant of p342E
(73). In particular, pCVSVEHBV was obtained as follows: The 540 bp
HindIII-HindIII fragment encompassing the SV40 origin of replication (74)
was ligated into plasmid pML (75) between the EcoRI site and the HindIII
site. The plasmid EcoRI site and SV40 HindIII site were made blunt by the
addition of Klenow DNA polymerase I in the presence of the 4 dNTPs prior
to digestion with HindIII. The resulting plasmid, pESV, was digested with
HindIII and BamHI and the 2900 b vector fragment isolated. To this
fragment was ligated a HindIII-BglII fragment of 2025 b from HBV modified
to contain a polylinker (DNA fragment containing multiple restriction
sites) at the EcoRI site. The HBV fragment encompasses the surface antigen
gene and is derived by EcoRI-BglII digestion of cloned HBV DNA (74). The
double stranded linker DNA fragment (5'dAAGCTTATCGATTCTAGAATTC3' . . . )
was digested with HindIII and EcoRI and added to the HBV fragment,
converting the EcoRI-BglII fragment to a HindlII-BglII fragment. Although
this could be done as a 3 part ligation consisting of linker, HBV
fragment, and vector, it is more convenient and was so performed to first
add the HindIII-EcoRI linker to the cloned HBV DNA and then excise the
HindIII-BglII fragment by codigestion of the plasmid with those enzymes.
The resulting plasmid, pCVSVEHBV, contains a bacterial origin of
replication from the pBR322 derived pML, and ampicillin resistance marker,
also from pML, an SV40 fragment oriented such that the early promoter will
direct the transcription of the inserted HBV fragment, and the surface
antigen gene from HBV. The HBV fragment also provides a polyadenylation
signal for the production of polyadenylated mRNAs such as are normally
formed in the cytoplasm of mammalian cells.!
The plasmid pCVSVEHBV contained a useful ClaI site immediately 5' to an
XbaI site in the polylinker. This plasmid was opened with XbaI and BamHI
(in the Hepatitis Ag 3' untranslated region) and the ends were filled in
with DNA polymerase. This removed the Hepatitis surface antigen coding
region but retained its 3' polyadenylation signal region, as well as the
SV40 promoter. Into this vector was ligated a 1883 b EcoRI fragment (with
filled in ends) from the cDNA clone .lambda.10.3. This contained the final
77 coding base pairs of Factor VIII, the 1805 b 3' untranslated region, 8
adenosine residues, and the filled in EcoRI adaptor. By virtue of joining
the filled in restriction sites, the EcoRI end was recreated at the 5' end
(from filled in XbaI joined to filled in EcoRI) but destroyed at the 3'
end (filled in EcoRI joined to filled in BamHI). This plasmid was called
pCVSVE/10.3.
The complete factor VIII cDNA region was joined in a three-piece ligation.
pCVSVE/10.3 was opened with ClaI and EcoRI and served as vector for the
insertion of the 3870 b ClaI/ScaI fragment from pF8Cla-Sca and the 3182 b
ScaI/EcoRI fragment from pF8Sca-RI. This expression plasmid was called
pSVEFVIII.
b. Construction for Expression of Factor VIII in Tissue Culture Cells
A variant vector based on PSVEFVIII, containing the adenovirus major late
promoter, tripartide leader sequence, and a shortened Factor VIII
3'-untranslated region produced active factor VIII when stably transfected
into BHK cells.
FIG. 12 shows the construction of pAML3P.8cl, the expression plasmid that
produces active factor VIII. To make this construction first the SstII
site in pFD11 (49r) and the ClaI site in pEHED22 (49y) were removed with
Klenow DNA polymerase I. These sites are in the 3' and 5' untranslated
regions of the DHFR gene on these plasmids. Then a three-part ligation of
fragments containing the deleted sites and the hepatitis B surface antigen
gene from pCVSVEHBS (supra) was performed to generate the vector
pCVSVEHED22.DELTA.CS which has only one ClaI and one SstII site. The
plasmid pSVEFVIII containing the assembled factor VIII gene (FIG. 11) was
cleaved with ClaI and HpaI to excise the entire coding region and about
380 b of the 3' untranslated region. This was inserted into the ClaI,
SstII deletion vector at its unique ClaI and HpaI sites, replacing the
surface antigen gene to give the expression plasmid pSVE.8c1D.
Separately, the adenovirus major late promoter with its tripartite 5'
leader was assembled from two subclones of portions of the adenovirus
genome along with a DHFR expression plasmid, pEHD22 (49y). Construction of
the two adenovirus subclones, pUCHSX and pMLP2, is described in the
methods. pMLP2 contains the SstI to HindIII fragment from adenovirus
coordinates 15.4 to 17.1 cloned in the SstI to HindIII site of pUC13 (59).
pUCHSX contains the HindIII to XhoI fragment coordinates 17.1 to 26.5
cloned in the HindIII to SalI site of pUC13. When assembled at the HindIII
site, these two adenovirus fragments contain the major late promoter of
adenovirus, all of the first two exons and introns, and part of the third
exon up to the XhoI site in the 5' untranslated region.
A three-part ligation assembled the adenovirus promoter in front of the
DHFR gene in the plasmid pAML3P.D22. This put a ClaI sit shortly following
the former XhoI site in the third exon of the adenovirus tripartite 5'
leader. Finally, the SV40 early promoter of the factor VIII expression
plasmid, pSVE.8c1D, was removed with ClaI and SalI and replaced with the
adenovirus promoter to generate the final expression plasmid, pAML3P.8c1
(see FIG. 12). This plasmid contains the adenovirus tripartite leader
spliced in the third exon to the 5' untranslated region of factor VIII.
This is followed by the full length Factor VIII structural gene including
its signal sequence. The 3' untranslated region of the factor VIII gene is
spliced at the HpaI site to the 3' untranslated region of Hepatitis B
surface antigen gene. This is followed by the DHFR gene which has an SV40
early promoter and a Hepatitis 3' untranslated region conferring a
functional polyadenylation signal.
The factor VIII expression plasmid, pAML3P.8c1, was cotransfected into BHK
cells with the neomycin resistance vector pSVEneoBal6 (ATCC No. CRL 8544,
deposited 20 April 1984). These cells were first selected with G418
followed by a selection with methotrexate.
Initial characterization of the Factor VIII RNA produced by the BHK cell
line was performed by Northern analysis of poly(A).sup.+ cytoplasmic RNA
by hybridization to a .sup.32 P-labeled Factor VIII DNA probe. This
analysis shows a band approximately 9 kb in length. Based on hybridization
intensities, this band is about 100 to 200 fold enriched when compared to
the 9 kb band found in the CH-2 cell line.
9. Identification of Recombinant Factor VIII
a. Radioimmune assay
Radioimmune assays were performed as described in the Methods on
supernatants and lysed cells from the BHK Factor VIII producing cell line.
Table 1 shows that the supernatants (which contain factor VIII activity)
(See 96) also contain approximately equal amounts of the 210 kD (C10) and
the 80 kD (C7F7) portions of Factor VIII as judged by these RIAs. Factor
VIII can also be detected in the cell lysates by both RIAs. Control cell
lines not expressing factor VIII produced RIA values of less than 0.001
units per ml.
TABLE 1
______________________________________
Factor VIII RIA of BHK cell line transfected with pAML3P.8cl
C10 C7F7
______________________________________
Cell Supernatant
Exp. 1 0.14 U/ml 0.077
Exp. 2 0.022 0.021
Cell Lysate 0.42 0.016
Exp. 1
______________________________________
I.sup.125 cpm bound were converted to units/ml with a standard curve based
on dilutions of normal plasma. All values are significantly above
background. Limits of detection were 0.005 U/ml for the CIO and 0.01 U/ml
for the C7F7 assays.
b. Chromogenic Assay on BHK Cell Media
As is shown in Table 2, media from these cells generated an absorbance at
405 nm when tested in the Coatest assay. As described above, this assay is
specific for factor VIII activity in the activation of factor X. Addition
of monoclonal antibodies specific for factor VIII decreased the amount of
factor X.sub.a generated as evidenced by the decrease in absorbance from
0.155 for the media to 0.03 for the media plus antibodies (after
subtracting out the blank value). Therefore, the cells are producing an
activity which functions in an assay specific for factor VIII activity and
this activity is neutralized by antibodies specific for factor VIII.
Incubation of the media in the reaction mixture without the addition of the
factor 1X.sub.a, factor X, and phospholipid did not result in an increase
in the absorbance at 405 nm above the blank value. The observed activity
is therefore not due to the presence of a nonspecific protease cleaving
the substrate, and in addition neutralized by antibodies specific for
factor VIII.
TABLE 2
______________________________________
Factor VIII Activity of BHK cell line determined by chromogenic
assay.sup.1.
Absorbance
Absorbance
at 405 nm
at (Control value
Sample 405 nm subtracted).
______________________________________
Media 0.193 0.155
Buffer control.sup.2
0.038 (0.0)
Media + factor VIII antibody.sup.3
0.064 0.030
Buffer control.sup.2 + factor VIII antibody
0.034 (0.0)
______________________________________
.sup.1 Reactions modified as follows: 50 .mu.l each of IX.sub.a
/X/phospholipid, CaCl.sub.2, and 1100 diluted sample were incubated 10
minutes at 37.degree. C. S2222 (50 .mu.l was added and reaction terminate
with 100 .mu.l) of 50 percent acetic acid after 60 minutes at 37.degree.
C.
.sup.2 Buffer used in place of sample was 0.05M Tris HC1, pH 7.3,
containing 0.2 percent bovine serum albumin.
.sup.3 Antibody was a mixture of C8 and C7F7 (10 .mu.g each). The media
was preincubated 5 minutes prior to start of assay.
c. Chromatography of Media on Monoclonal Resin
Serum containing media containing factor VIII activity was chromatographed
on the C8 monoclonal antibody (ATCC No. 40115, deposited 20 April 1984)
column as described (supra). The eluted fractions were diluted 1:100 and
assayed for activity. To 50 .mu.l of the diluted peak fraction was added
various monoclonal antibodies known to be neutralizing for plasma factor
VIII activity. The results shown in Table 3 demonstrate that the factor
VIII activity eluted from the column (now much more concentrated than the
media) was also neutralized by these factor VIII antibodies.
TABLE 3
______________________________________
Chromogenic Assay.sup.1 of Peak Fraction of Monoclonal Antibody Eluate
Absorbance at
Sample 405 nM.sup.1
______________________________________
Peak Fraction.sup.2 0.186
Peak fraction plus factor VIII Antibody.sup.3
0.060
Buffer Control 0.000
Buffer Control plus factor VIII Antibody
0.045
______________________________________
.sup.1 Assay was perfomed as follows: 50 .mu.l of diluted sample was
incubated 5 minutes with 50 .mu.l of IX.sub.a /X/phospholipid solution at
37.degree. C. The reaction was incubated with 50 .mu.l of CaCl.sub.2, and
allowed to proceed 10 minutes at 37.degree. C. The chromogenic substrate
(50 .mu.l) was added, and the reaction teminated by the addition of 100
.mu.l of 50 percent acetic acid after 10 minutes.
.sup.2 Peak fraction was diluted 1:100 in 0.05M Tris, pH 7.2, containing
0.15M NaCl for assays.
.sup.3 Antibody was 10 .mu.l of Symbiotic antibody added to the diluted
sample and incubated 5 minutes at room temperature.
d. Coagulant Activity of Purified Factor VIII.
The activity detected in the cell media was purified and concentrated by
passage over a C8 monoclonal resin (supra). The peak fraction was dialyzed
against 0.05M imidazole, pH6.9, containing 0.15M NaCl, 0.02M glycine ethyl
ester. 0.01M CaCl.sub.2, and 10 percent glycerol in order to remove the
elution buffer. The
Activity peak fraction was assayed by coagulation analysis in factor VIII
deficient plasma (Table 4). A fibrin clot was observed at 84 seconds. With
no addition, the hemophilia plasma formed a clot in 104.0 seconds.
Therefore, the eluted fraction corrected the coagulation defect in
hemophilia plasma. Normal human plasma was diluted and assayed in the same
manner. A standard curve prepared from this plasma indicated that the
eluted fraction had approximately 0.01 units per milliter of factor VIII
coagulant activity.
TABLE 4
______________________________________
Coagulant Activity of Monoclonal Antibody Purified Factor VIII
Sample Clotting time (sec.)
______________________________________
Recombinant factor VIII.sup.1a
86.5
Recombinant factor VIII.sup.1a and C7F7 antibody
101.3
Control.sup.2 101.3
Recombinant factor VIII.sup.1b
82.4
Recombinant factor VIII.sup.1b
110.6
and 10.lambda. Synbiotic antibody
Control.sup.2 95.5
______________________________________
.sup.1 Factor VIII was peak fraction eluted from the C8 monoclonal resin
and dialyzed for 11/2 (1a) or 2 (1b) hours in order to remove elution
buffer.
.sup.2 Control buffer was 0.05M Tris, pH 7.3, containing 0.2 percent
bovine serum albumin.
e. Thrombin Activation of Purified Factor VIII.
Activation of coagulant activity by thrombin is a well established property
of factor VIII. The eluted fraction from the monoclonal column was
analyzed for this property. After dialysis of the sample to remove the
elution buffer (supra), 100 .mu.l of the eluate was diluted with 100 .mu.l
of 0.05M imidazole, pH7.6, containing 0.15M NaCl, 0.02M glycine ethyl
ester, 0.01M CaCl.sub.2 and 10 percent glycerol. This dilution was
performed to dilute further any remaining elution buffer (which might
interfere with thrombin functioning) as well as to increase the pH of the
reaction mixture. Thrombin (25 ng) was added to the solution and the
reaction was performed at room temperature. Aliquots of 25 .mu.l were
removed at various time points, diluted 1:3, and assayed for coagulation
activity. The results are shown in FIG. 17. The factor VIII activity
increased with time, and subsequently decreased, as expected for a factor
VIII activity. The amount of thrombin added did not clot factor VIII
deficient plasma in times observed for these assays, and the observed time
dependent increase and subsequent decrease in observed coagulation time
proved that the activity being monitored was in fact due to thrombin
activation of factor VIII. The observed approximately 20-fold activation
by thrombin is in agreement with that observed for plasma factor VIII.
f. Binding of Recombinant Factor VIII to Immobilized von Willebrand
Sepharose.
Factor VIII is known to circulate in plasma in a reversible complex with
von Willebrand Factor (vWF) (10-20). A useful form of recombinant factor
VIII should therefore also possess this capacity for forming such a
complex in order to confirm identity as factor VIII. In addition, the
ability to form such a complex would prove the ability of a recombinant
factor VIII to form the natural, circulating form of the activity as the
factor VIII/vWF complex upon infusion into hemophiliacs. In order to test
the ability of recombinant factor VIII to interact with vWF, vWF was
purified and immobilized on a resin as follows:
Human von Willebrand factor was prepared by chromatography of human factor
VIII concentrates (purchased from, e.g., Cutter Laboratories) on a
Sepharose CL4B resin equilibrated with 0.05M Tris, pH 7.3, containing
0.15M NaCl. The von Willebrand factor elutes at the void volume of the
column. This region was pooled, concentrated by precipitation with
ammonium sulfate at 40 percent of saturation and re-chromatographed on the
column in the presence of the above buffer containing 0.25M CaCl.sub.2 in
order to separate the factor VIII coagulant activity from the von
Willebrand factor. The void volume fractions were again pooled,
concentrated using ammonium sulfate, and dialyzed against 0.1M sodium
bicarbonate. The resulting preparation was covalently attached to cyanogen
bromide activated Sepharose (purchased from Pharmacia) as recommended by
the manufacturer. The column was washed with 0.02M Tris, pH 7.3,
containing 0.05M NaCl and 0.25M CaCl.sub.2 in order to remove unbound
proteins. The recombinant factor VIII was prepared in serum free media and
applied to a 1.0 ml column of the vWF resin at room temperature.
The column was washed to remove unbound protein and eluted with 0.02M Tris,
pH 7.3, containing 0.05M NaCl and 0.25M CaCl.sub.2. Fractions of 1.0 ml
were collected, diluted 1:10 and assayed. The results are shown in Table
5. The factor VIII activity is absorbed from the media onto the column.
The activity can subsequently be eluted from the column using high salt
(Table 5), as expected for the human factor VIII. Therefore, the factor
VIII produced by the BHK cells has the property of specific interaction
with the von Willebrand factor protein.
TABLE 5
______________________________________
Absorbance
Sample at 405 nm
______________________________________
Cell Media 0.143
Wash 0.075
Eluted Fractions
1 0.000
2 0.410
3 0.093
4 0.017
5 0.000
6 0.000
______________________________________
.sup.1 Assay procedures were that recommended by the manufacturer, except
that all volumes were decreased by one half.
10. Analysis of Fusion Proteins
The purpose of this set of experiments was to prove immunological identity
of the protein encoded by the clone with the polypeptides in plasma. This
was accomplished by expressing portions of the gene as fusion proteins in
E. coli. All or part of the coding sequences of the cloned gene can be
expressed in forms designed to provide material suitable for raising
antibodies. These antibodies, specific for desired regions of the cloned
protein, can be of use in analysis and purification of proteins. A series
of E. coli/factor VIII "fusion proteins" were prepared for this purpose.
Fragments of factor VIII clones were ligated into the BglII site of the
plasmid pNCV (70) in such a way as to join factor VIII coding sequences,
in proper reading frame, to the first 12 amino acids of the fused E. coli
trp LE protein (48, 70, 71). Substantial amounts of recombinant protein
product are usually produced from this strong trp promoter system.
pfus1 was constructed by isolating a 189 bp StuI/HincII fragment of factor
VIII (coding for amino acids 1799-1860) and ligating this into the SmaI
site of pUC13 (49K). This intermediate plasmid was digested with BamHI and
EcoRI and the 200 bp fragment inserted into pNCV (70) from which the 526
bpBglII to EcoRI fragment had been removed. This plasmid, pfus1, produces
under trp promoter control a 10 kD fusion protein consisting of 16 trpLE
and linker coded amino acids, followed by 61 residues of factor VIII and a
final 9 linker coded and trpE carboxy terminal residues.
pfus3 was constructed by removing a 290 bp AvaII fragment of factor VIII
(amino acids 1000-1096), filling in the overhanging nucleotides using
Klenow fragment of DNA polymerase, and ligating this now blunt-ended DNA
fragment into pNCV which had been cut with BglII and similarly filled in.
This plasmid, with the filled in fragment in the proper orientation (as
determined by restriction digests and DNA sequence analysis), directs the
synthesis of an approximately 40 kD fusion protein containing 97 amino
acids of factor VIII embedded within the 192 amino acid trpLE protein.
pfus4 was made by cutting a factor VIII subclone, .lambda.222.8, with BanI,
digesting back the overhang with nuclease S.sub.1, followed by PstI
digestion and isolation of the resulting 525 bp blunt/PstI fragment (amino
acids 710-885). This was ligated into pNCV, which had been digested with
BglII, treated with S.sub.1, digested with PstI, and the vector fragment
isolated. pfus4 directs the synthesis of a 22 kD fusion protein containing
175 amino acids of factor VIII following the initial 12 amino acids of
trpLE.
The fusion proteins were purified and injected into rabbits in order to
generate antibodies as described Supra. These antibodies were tested for
binding to plasma derived factor VIII by Western Blot analysis.
The results of such a Western transfer are shown in FIG. 13. Each of the
fusion proteins reacts with the plasma factor VIII. Fusion 1 was generated
from the region of the gene encoding an 80,000 dalton polypeptide. It can
be seen that fusion 1 antisera react only with the 80,000 dalton band, and
do not react with the proteins of higher molecular weight. Fusion 3 and 4
antisera show cross reactivity with the proteins of greater than 80,000
daltons, and do not react with the 80,000 dalton band. The monoclonal
antibody C8 is an activity neutralizing monoclonal directed against factor
VIII and is known to react with the 210,000 dalton protein. FIG. 14
demonstrates that fusion 4 protein will react with this monoclonal
antibody, thereby demonstrating that the amino acid sequence recognized by
C8 is encoded by fusion 4 polypeptide. This further supports the identity
of fusion 4 protein containing protein sequences encoding the 210,000
dalton protein. The above studies conclusively prove that the gene encodes
the amino acid sequence for both the 210,000 and 80,000 dalton proteins.
11. Pharmaceutical Compositions
The compounds of the present invention can be formulated according to known
methods to prepare pharmaceutically useful compositions, whereby the human
factor VIII product hereof is combined in admixture with a
pharmaceutically acceptable carrier vehicle. Suitable vehicles and their
formulation, inclusive of other human proteins, e.g. human serum albumin,
are described for example in Remington's Pharmaceutical Sciences by E. W.
Martin, which is hereby incorporated by reference. Such compositions will
contain an effective amount of the protein hereof together with a suitable
amount of vehicle in order to prepare pharmaceutically acceptable
compositions suitable for effective administration to the host. For
example, the human factor VIII hereof may be parenterally administered to
subjects suffering, e.g., from hemophilia A.
The average current dosage for the treatment of a hemophiliac varies with
the severity of the bleeding episode. The average doses administered
intraveneously are in the range of: 40 units per kilogram for
pre-operative indications, 15 to 20 units per kilogram for minor
hemorrhaging, and 20 to 40 units per kilogram administered over an 8-hours
period for a maintenance dose.
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